Synthesis and novel salt forms of (r)-3-((e)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridine

ABSTRACT

The present invention relates to (R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridine, its salt forms, and to processes for the commercial-scale production of these compounds in sufficient purity and quality for use in pharmaceutical compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.13/125,892 filed Jul. 19, 2011, which is a 371 of internationalapplication no. PCT/US2009/066092 filed Nov. 30, 2011, which claimsbenefit to U.S. provisional application No. 61/118,885 filed Dec. 1,2008, each of which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to compounds that bind to and modulate theactivity of neuronal nicotinic acetylcholine receptors, to novel saltsthereof, to processes for preparing these compounds, to pharmaceuticalcompositions containing these compounds, and to methods of using thesecompounds for treating a wide variety of conditions and disorders,including those associated with dysfunction of the central nervoussystem (CNS).

BACKGROUND OF THE INVENTION

The therapeutic potential of compounds that target neuronal nicotinicreceptors (NNRs), also known as nicotinic acetylcholine receptors(nAChRs), has been the subject of several reviews. See, for example,Breining et al., Ann. Rep. Med. Chem. 40: 3 (2005), Hogg and Bertrand,Curr. Drug Targets: CNS Neurol. Disord. 3: 123 (2004). Among the kindsof indications for which NNR ligands have been proposed as therapies areCNS disorders mentioned below. There exists a heterogeneous distributionof nAChR subtypes in both the central and peripheral nervous systems.For instance, the nAChR subtypes which are predominant in vertebratebrain are α4β2, α7, and α3β2, whereas those which predominate at theautonomic ganglia are α3β4 and those of neuromuscular junction areα1β1γ6 and α1β1γε.

A limitation of some nicotinic compounds is that they are associatedwith various undesirable side effects due to non-specific binding tomultiple nAChR subtypes. For example, binding to and stimulation ofmuscle and ganglionic nAChR subtypes can lead to side effects which canlimit the utility of a particular nicotinic binding compound as atherapeutic agent.

The commercial development of a drug candidate involves many steps,including the development of a cost effective synthetic method that isadaptable to a large scale manufacturing process. Commercial developmentalso involves research regarding salt forms of the drug substance thatexhibit suitable purity, chemical stability, pharmaceutical properties,and characteristics that facilitate convenient handling and processing.Furthermore, compositions containing the drug substance should haveadequate shelf life. That is, they should not exhibit significantchanges in physicochemical characteristics such as, but not limited to,chemical composition, water content, density, hygroscopicity, stability,and solubility upon storage over an appreciable period of time.Additionally, reproducible and constant plasma concentration profiles ofdrug upon administration to a patient are also important factors.

Solid salt forms are generally preferred for oral formulations due totheir tendency to exhibit these properties in a preferential way; and inthe case of basic drugs, acid addition salts are often preferred salt.However, different salt forms vary greatly in their ability to impartthese properties and such properties cannot be predicted with reasonableaccuracy. For example, some salts are solids at ambient temperatures,while other salts are liquids, viscous oils, or gums at ambienttemperatures. Furthermore, some salt forms are stable to heat and lightunder extreme conditions and others readily decompose under much milderconditions. Salts also vary greatly in their hygroscopicity, the lesshygroscopic being more advantageous. Thus, the development of a suitableacid addition salt form of a basic drug for use in a pharmaceuticalcomposition is a highly unpredictable process.

Racemic5-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridine,its synthesis, and its hemi-galactarate salt form are disclosed in WO04/078752 which is incorporated by reference, and its counterparts.Because of the advantageous pharmacological properties of a singleenantiomer over its racemate, there is a need for a stereospecificsynthesis, preferably a process suitable for large-scale production.Furthermore, there is a need for salt forms that display improvedproperties, such as for example purity, stability, solubility, andbioavailability. Preferential characteristics of these novel salt formsinclude those that would increase the ease or efficiency of manufactureof the active ingredient and its pharmaceutical composition into acommercial drug product and improved stability of the drug over aprolonged period of time.

SUMMARY OF THE INVENTION

One aspect of the present invention is(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineor a pharmaceutically acceptable salt thereof. One aspect of the presentinvention is(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridinemono-L-malate or a hydrate or solvate thereof. Another aspect is(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridinehemi-galactarate or a hydrate or solvate thereof. Another aspect is(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineoxalate or a hydrate or solvate thereof. Another aspect is(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridinedi-p-toluoyl-D-tartrate or a hydrate or solvate thereof.

One aspect of the present invention is(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineor a pharmaceutically acceptable salt thereof substantially free of(S)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridine.In one embodiment,(S)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineis present in an amount of less than 25% by weight. In one embodiment,(S)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineis present in an amount of less than 15% by weight. In one embodiment,(S)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineis present in an amount of less than 5% by weight. In one embodiment,(S)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineis present in an amount of less than 2% by weight. In one embodiment,(S)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineis present in an amount of less than 1% by weight. In one embodiment,the(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineis free of a significant amount of(S)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridine.

One aspect of the present invention is a pharmaceutical compositioncomprising a compound as herein disclosed and one or morepharmaceutically acceptable adjuvant, carrier, or excipient. In oneembodiment, the pharmaceutical composition further comprises one or moreadditional therapeutic agent.

One aspect of the present invention includes a compound(R)-3-((E)-2-(Pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridinemono-L-malate or hemi-galactarate or oxalate or di-p-toluoyl-D-tartratefor use as a medicament in treating a NNR mediated disorder.

Another aspect includes a method for the treatment or prevention of aNNR mediated disorder comprising administering to a mammal in need ofsuch treatment, a therapeutically effective amount of(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineor a pharmaceutically acceptable salt thereof.

Another aspect includes use of a compound(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineor a pharmaceutically acceptable salt thereof in the preparation of amedicament for the treatment of a NNR mediated disorder.

In one embodiment of the aforementioned compound, method, or use, thedisorder is selected from the group consisting of CNS disorders,inflammation, inflammatory response associated with bacterial and/orviral infection, pain, metabolic syndrome, autoimmune disorders. In oneembodiment, the CNS disorder is selected from cognitive dysfunction inschizophrenia (CDS), Alzheimers Disease (AD), attention deficit disorder(ADD), pre-senile dementia (early onset of Alzheimer's Disease),dementia of the Alzheimer's type, mild cognitive impairment, ageassociated memory impairment and attention deficit hyperactivitydisorder (ADHD).

Another aspect of the present invention includes an administrationregimen of a pharmaceutical composition comprising administering(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridine,or a pharmaceutically acceptable salt thereof in amounts of between 7 to2200 μg/kg.

In the aspects and embodiments, another embodiment includes where the(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineis provided as the mono-L-malate, hemi-galactarate, oxalate, ordi-p-toluoyl-D-tartrate salt thereof.

Another aspect includes novel intermediates, including diethyl(R)-2-(1-(tert-butoxycarbonyl)pyrrolidin-3-yl)malonate;(R)-2-(1-(tert-butoxycarbonyl)pyrrolidin-3-yl)malonic acid; tert-butyl(R)-3-(2-hydroxyethyl)pyrrolidine-1-carboxylate; and tert-butyl(R)-3-(2-iodoethyl)pyrrolidine-1-carboxylate.

Another aspect includes a method of making(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridinethrough the intermediacy of one or more of diethyl(R)-2-(1-(tert-butoxycarbonyl)pyrrolidin-3-yl)malonate,(R)-2-(1-(tert-butoxycarbonyl)pyrrolidin-3-yl)malonic acid, tert-butyl(R)-3-(2-hydroxyethyl)pyrrolidine-1-carboxylate, and tert-butyl(R)-3-(2-iodoethyl)pyrrolidine-1-carboxylate.

Another aspect includes a method of making tert-butyl(R)-3-vinylpyrrolidine-1-carboxylate through the intermediacy of one ormore of diethyl (R)-2-(1-(tert-butoxycarbonyl)pyrrolidin-3-yl)malonate,(R)-2-(1-(tert-butoxycarbonyl)pyrrolidin-3-yl)malonic acid, tert-butyl(R)-3-(2-hydroxyethyl)pyrrolidine-1-carboxylate, and tert-butyl(R)-3-(2-iodoethyl)pyrrolidine-1-carboxylate.

Another aspect includes a method of purifying(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridine,with respect to isomeric(R)-3-((Z)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineand(R)-3-(1-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridine, byconversion to the oxalate salt and re-generation of the free base.

Combinations of aspects and embodiments form further embodiments of thepresent invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts novel object recognition (NOR) vs. dose for(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineor a salt thereof, hereinafter referred to as Compound A. Astatistically significant effect was observed for doses as low as 0.004μM/kg.

FIG. 2 depicts novel object recognition (NOR) vs. time for(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineor a salt thereof, Compound A, a dose at 0.004 μM/kg. A statisticallysignificant effect was observed out to 8 h after dosing.

FIG. 3 depicts results of Radial Arm Maze (RAM) Studies in which(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineor a salt thereof, Compound A, overcomes scopolamine induced deficits inthe radial arm maze.

DETAILED DESCRIPTION Definitions

The following definitions are meant to clarify, but not limit, the termsdefined. If a particular term used herein is not specifically defined,such term should not be considered indefinite. Rather, terms are usedwithin their accepted meanings.

As used herein, the term “compound(s)” may be used to mean the free baseform, or alternatively, a salt form of(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridine(Formula I), depending on the context, which will be readily apparent.Those skilled in the art will be able to distinguish the difference.

As used herein, the phrase “pharmaceutically acceptable” refers tocarrier(s), diluent(s), excipient(s) or salt forms of the compound ofFormula I that are compatible with the other ingredients of thecomposition and not deleterious to the recipient of the pharmaceuticalcomposition.

As used herein, the phrase “pharmaceutical grade” refers to a compoundor composition of a standard suitable for use as a medicine. Withreference to the discussion herein, pharmaceutical grade compounds ofthe present invention, particularly salt forms thereof, displayappropriate properties, including purity, stability, solubility, andbioavailability for use in a drug product. Preferential characteristicsinclude those that would increase the ease or efficiency of manufactureof the active ingredient and its composition into a commercial drugproduct. Furthermore, pharmaceutical grade compounds of the presentinvention may be synthesized using a stereospecific synthesis that isscalable to a large-scale production, namely displaying adequate purityand yield.

As used herein, the term “pharmaceutical composition” refers to acompound of the present invention optionally admixed with one or morepharmaceutically acceptable carriers, diluents, or excipients.Pharmaceutical compositions preferably exhibit a degree of stability toenvironmental conditions so as to make them suitable for manufacturingand commercialization purposes.

As used herein, the terms “effective amount”, “therapeutic amount”, or“effective dose” refer to an amount of the compound of the presentinvention sufficient to elicit the desired pharmacological ortherapeutic effects, thus resulting in effective prevention or treatmentof a disorder. Prevention of the disorder may be manifested by delayingor preventing the progression of the disorder, as well as the onset ofthe symptoms associated with the disorder. Treatment of the disorder maybe manifested by a decrease or elimination of symptoms, inhibition orreversal of the progression of the disorder, as well as any othercontribution to the well being of the patient.

As will be discussed in more detail below and with reference to FIGS. 1and 2, a statistically significant effect is observed for doses of thecompound of Formula I, or a pharmaceutically acceptable salt thereof, aslow as 0.004 μM/kg, including effects observed out to 8 h after dosing.The effective dose can vary, depending upon factors such as thecondition of the patient, the severity of the symptoms of the disorder,and the manner in which the pharmaceutical composition is administered.Thus, as used herein, the effective dose may be less than 100 mg, inanother embodiment less than 50 mg, in another embodiment less than 10mg, or in another embodiment less than 1 mg. These effective dosestypically represent the amount administered as a single dose, or as oneor more doses administered over a 24 h period.

As used herein, the phrase “substantially’ or ‘sufficiently’ quality,purity or pure, includes greater than 20%, preferably greater than 30%,and more preferably greater than 40% (e.g. greater than any of 50, 60,70, 80, or 90%) quality or purity.

The term “stability” as defined herein includes chemical stability andsolid state stability, where the phrase “chemical stability” includesthe potential to store salts of the invention in an isolated form, or inthe form of a pharmaceutical composition in which it is provided inadmixture with pharmaceutically acceptable carriers, diluents,excipients, or adjuvants, such as in an oral dosage form, such as atablet, capsule, or the like, under normal storage conditions, with aninsignificant degree of chemical degradation or decomposition, and thephrase “solid state stability”, includes the potential to store salts ofthe invention in an isolated solid form, or in the form of a solidpharmaceutical composition in which it is provided in admixture withpharmaceutically acceptable carriers, diluents, excipients, oradjuvants, such as in an oral dosage form, such as a tablet, capsule, orthe like, under normal storage conditions, with an insignificant degreeof solid state transformation, such as crystallization,recrystallization, solid state phase transition, hydration, dehydration,solvation, or desolvation.

Examples of “normal storage conditions” include one or more oftemperatures of between −80° C. and 50° C., preferably between 0° C. and40° C. and more preferably ambient temperatures, such as 15° C. to 30°C., pressures of between 0.1 and 2 bars, preferably at atmosphericpressure, relative humidity of between 5 and 95%, preferably 10 to 60%,and exposure to 460 lux or less of UV/visible light, for prolongedperiods, such as greater than or equal to six months. Under suchconditions, salts of the invention may be found to be less than 5%, morepreferably less than 2%, and especially less than 1%, chemicallydegraded or decomposed, or solid state transformed, as appropriate. Theskilled person will appreciate that the above-mentioned upper and lowerlimits for temperature, pressure, and relative humidity representextremes of normal storage conditions, and that certain combinations ofthese extremes will not be experienced during normal storage (e.g. atemperature of 50° C. and a pressure of 0.1 bar).

As used herein, the term “disorder”, unless stated otherwise, means anycondition, dysfunction, or disease associated with NNR receptoractivity.

Compounds

One embodiment of the present invention relates to(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridine(Formula I) or a pharmaceutically acceptable salt thereof.

As will be appreciated by those skilled in the art, different namingconventions may name a compound differently. Thus, Compound A may benamed(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineor, alternatively,(R)-3-(2-pyrrolidin-3-yl)-vinyl)-5-((tetrahydro-2H-pyran-4-yl)oxy)pyridine.Such naming conventions should not be used to introduce ambiguity tothis specification.

In one embodiment, the compound of Formula I or a pharmaceuticallyacceptable salt thereof is substantially pure. In one embodiment, thecompound of Formula I or a pharmaceutically acceptable salt thereof issubstantially free of(S)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridine.In one embodiment, the compound of Formula I or a pharmaceuticallyacceptable salt thereof is present in an amount of about 75% by weightcompared to(S)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridine,preferably greater than 85% by weight, more preferably greater than 95%by weight, more preferably greater than 98% by weight, and mostpreferably 99% by weight or greater. One embodiment relates to 100% pure(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridine(Formula I).

Process

One embodiment of the present invention relates to a method for thepreparation of(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineor a pharmaceutically acceptable salt thereof substantially free of(S)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineby weight. Another embodiment of the present invention relates to amethod for the preparation of(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineor a pharmaceutically acceptable salt thereof containing less than 25%,preferably less than 15%, more preferable less than 5%, even morepreferably less than 2%, and most preferably less than 1% of(S)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineby weight, without the use of a chiral chromatographic separation step.In one embodiment of the present invention, a method for the manufactureof substantially pure(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineis provided, without reliance upon chromatographic separation.

General Synthetic Methods

Racemic3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridine canbe synthesized as reported in PCT WO2004/078752, herein incorporated byreference, using a palladium catalyzed coupling of tert-butyl3-vinylpyrrolidine-1-carboxylate with the3-bromo-5-(tetrahydro-2H-pyran-4-yloxy)pyridine, followed by removal ofthe tert-butoxycarbonyl protecting group. In the racemic synthesis, therequisite tert-butyl 3-vinylpyrrolidine-1-carboxylate was produced bytreating tert-butyl 3-formylpyrrolidine-1-carboxylate withmethylenetriphenylphosphorane (Wittig reagent). While tert-butyl3-formylpyrrolidine-1-carboxylate can be made by several methods, it wasnot an ideal intermediate for a single enantiomer synthesis, in that itis susceptible to racemization during the Wittig reaction. Thus, a newsynthetic route, one characterized by stereochemical fidelity, wasdevised.

The compounds may be prepared according to the following methods usingcommercially available starting materials and reagents.

(R)-3-((E)-2-(Pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridinemay be prepared via palladium catalyzed coupling of tert-butyl(R)-3-vinylpyrrolidine-1-carboxylate (compound 9) and3-bromo-5-(tetrahydro-2H-pyran-4-yloxy)-pyridine (compound 12) asoutlined in Scheme 3.

The preparation of compound 9 is outlined in Scheme 1. Commerciallyavailable tert-butyl (R)-3-hydroxypyrrolidine-1-carboxylate (compound 1)is treated with methanesulfonyl chloride to give tert-butyl(R)-3-(methylsulfonyloxy)pyrrolidine-1-carboxylate (compound 2), whichthen is reacted with diethylmalonate and a suitable base (e.g.,potassium tert-butoxide or sodium ethoxide) to give diethyl(R)-2-(1-(tert-butoxycarbonyl)pyrrolidin-3-yl)malonate (compound 3) withinverted stereochemistry around the chiral carbon.

Suitable solvents for these reactions may be selected from the group oftoluene, xylenes, 1-methyl-2-pyrrolidinone, dimethylformamide,dimethylacetamide, ethanol, tert-butanol, tetrahydrofuran,1,2-dimethoxyethane, dioxane, and mixtures thereof. In one embodimentthe solvent for the methanesulfonic ester formation toluene, and thesolvent for the malonate displacement is 1-methyl-2-pyrrolidinone. Inanother embodiment the solvent for the malonate displacement is ethanol.Suitable bases for these reactions may be selected from the group oftriethylamine, diethylisopropylamine, diisopropylethylamine, potassiumtert-butoxide, sodium metal, sodium hydride, sodium ethoxide, potassiumhydride and lithium hydride. In one embodiment the base for themethanesulfonic ester formation is triethylamine, and the base for themalonate displacement is potassium tert-butoxide. In another embodimentthe base for the malonate displacement is sodium ethoxide.

Hydrolysis of diester 3 with aqueous potassium hydroxide yields(R)-2-(1-(tert-butoxycarbonyl)pyrrolidin-3-yl)malonic acid (compound 4),which is decarboxylated to afford(R)-2-(1-(tert-butoxycarbonyl)pyrrolidin-3-yl)acetic acid (compound 5).Suitable solvents for these reactions may be selected from the group ofwater, ethanol, tetrahydrofuran, dimethylformamide, dimethylacetamide,1,2-dimethoxyethane, dioxane, 1-methyl-2-pyrrolidinone, toluene,dimethylsulfoxide, and mixtures thereof. In one embodiment the solventfor the ester hydrolysis is aqueous tetrahydrofuran, and the solvent forthe decarboxylation is 1-methyl-2-pyrrolidinone. In another embodimentthe solvent for the ester hydrolysis is ethanol, and the solvent for thedecarboxylation is a mixture of dimethylsufloxide and toluene. Suitablebases for the hydrolysis reaction may be selected from the group ofpotassium hydroxide, sodium hydroxide, potassium carbonate, sodiumcarbonate, barium hydroxide and cesium carbonate. In one embodiment thebase is potassium hydroxide.

Reduction of compound 5 gives tert-butyl(R)-3-(2-hydroxyethyl)pyrrolidine-1-carboxylate (compound 6), which maybe reacted with methanesulfonyl chloride and then sodium iodide to givetert-butyl (R)-3-(2-(methylsulfonyloxy)ethyl)pyrrolidine-1-carboxylate(compound 7) and tert-butyl (R)-3-(2-iodoethyl)pyrrolidine-1-carboxylate(compound 8), respectively. Suitable solvents for the reduction reactionmay be selected from the group of tetrahydrofuran, ether, dioxane,1,2-dimethoxyethane, and mixtures thereof. In one embodiment the solventis tetrahydrofuran. Suitable reducing agents may be selected from thegroup of borane, diborane, borane-tetrahydrofuran complex,borane-dimethyl ether complex and borane-dimethylsulfide complex.

Suitable solvents for the methanesulfonic ester formation may beselected from the group of toluene, xylenes, ether, tetrahydrofuran,1,2-dimethoxyethane, dioxane, and mixtures thereof. In one embodimentthe solvent for the methanesulfonic ester formation is toluene. Suitablebases for the methanesulfonic ester formation may be selected from thegroup of triethylamine, diethylisopropylamine and diisopropylethylamine.In one embodiment the base for the methanesulfonic ester formation istriethylamine. Suitable solvents for the iodide displacement may beselected from the group of 1-methyl-2-pyrrolidinone, dimethylformamide,dimethylacetamide, ethanol, tert-butanol, tetrahydrofuran,1,2-dimethoxyethane, dioxane, dimethylsulfoxide, and mixtures thereof.In one embodiment the solvent for the iodide displacement is1,2-dimethoxyethane.

Finally, treatment of compound 8 with potassium tert-butoxide gives ofcompound 9. Suitable solvents for this reaction may be selected from thegroup of 1,2-dimethoxyethane, 1-methyl-2-pyrrolidinone,dimethylformamide, dimethylacetamide, ethanol, tetrahydrofuran, dioxaneand mixtures thereof. In one embodiment the solvent is1,2-dimethoxyethane. Suitable bases for this reaction may be selectedfrom the group of potassium tert-butoxide, sodium ethoxide anddiazabicycloundecane. In another embodiment the base is potassiumtert-butoxide.

One embodiment of the invention relates to a process for the preparationof compound 9 using the reaction steps as outlined in Scheme 1 and inthe discussion above.

The preparation of 3-bromo-5-(tetrahydro-2H-pyran-4-yloxy)-pyridine(compound 12) is outlined in Scheme 2. Coupling of3-bromo-5-hydroxypyridine (compound 10) with4-hydroxytetrahydro-2H-pyran (compound 11) gives compound 12. Suitableconditions for the coupling include those in which a phosphine (e.g.,triphenylphosphine) and an azo compound (e.g., diethyl azodicarboxylate,also known as DEAD) are used, in an inert solvent, to effect thecoupling (e.g., toluene). Alternately, other conditions, in which theoxo anion of 3-bromo-5-hydroxypyridine displaces a leaving group fromthe 4-position of tetrahydropyran, may be employed.

One embodiment of the invention relates to a process for the preparationof compound 12 using the reaction steps as outlined in Scheme 2 andabove.

The final steps in the preparation of(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridine(free base form) is illustrated in Scheme 3. Compounds 9 and 12 arecoupled via a palladium acetate mediated coupling reaction to affordtert-butyl(R)-(E)-3-(2-(5-(tetrahydro-2H-pyran-4-yloxy)pyridin-3-yl)vinyl)pyrrolidine-1-carboxylate(also known as(R)-5-(1-(tert-butoxycarbonyl)-(E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridine,compound 13), which is de-protected to give(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridine(compound 14). Suitable solvents for the palladium-catalyzed couplingreaction may be selected from the group of 1-methyl-2-pyrrolidinone,dimethylformamide, dimethylacetamide and acetonitrile. In one embodimentthe solvent is 1-methyl-2-pyrrolidinone. Suitable bases for thepalladium catalyzed coupling reaction may be selected from the group oftriethylamine, diethylisopropylamine, diisopropylethylamine. In oneembodiment the base is diisopropylethylamine. Suitable phosphine ligandsfor the palladium catalyzed coupling reaction may be selected from thegroup of tri-n-butylphosphine, tri-tert-butylphosphine,tricyclohexylphosphine, triphenylphosphine and tri-o-tolylphosphine. Inone embodiment the phosphine ligand is tricyclohexylphosphine. Suitablepalladium catalysts for the palladium catalyzed coupling reaction may beselected from the group of palladium acetate, palladium chloride anddipalladium tris(dibenzylacetone). In one embodiment the palladiumcatalyst is palladium acetate. Suitable solvents for the de-protectionreaction may be selected from the group of water, dichloromethane,chloroform and dichloroethane. In one embodiment the solvent isdichloromethane. In another embodiment the solvent for the de-protectionreaction is water. Suitable acids for the de-protection reaction may beselected from the group of trifluoroacetic acid, hydrochloric acid andsulfuric acid. In one embodiment the acid is trifluoroacetic acid.

One embodiment of the invention relates to a process for the preparationof compound 14 using the reaction steps as outlined above in Schemes 1,2 and 3. The invention further relates to a process for the preparationof the salt form of(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridinemalate comprising the additional step of reacting the free base withL-malic acid in a mixture of 2-propanol and isopropyl acetate or othersuitable solvent as described below.

Another embodiment of the invention relates to the formation of theoxalate salt of compound 14 and the use of the oxalate salt as apurification intermediate in the production of compound 14. Thepalladium catalyzed coupling of compounds 9 and 12 produces a mixture ofmaterials in which compound 13 predominates, typically representing75-80% of the coupling products. The remaining coupling products includethe corresponding Z isomer, tert-butyl(R)—(Z)-3-(2-(5-(tetrahydro-2H-pyran-4-yloxy)pyridin-3-yl)vinyl)pyrrolidine-1-carboxylate,and a so-called “exo” isomer, tert-butyl(R)-3-(1-(5-(tetrahydro-2H-pyran-4-yloxy)pyridin-3-yl)vinyl)pyrrolidine-1-carboxylate,typically representing ˜5% and up to 20% of the coupling productsrespectively. Removal of these minor isomers from the major, desiredisomer was unexpectedly and conveniently accomplished by de-protectionof the mixture of isomers, followed by conversion of the free base tothe oxalate salt. Initial precipitation of the oxalate salt inwater/2-propanol mixtures, for example, gives compound 14 in which theisomeric impurities are reduced to <1% each or better. Furtherpurification can be accomplished by recrystallization.

Examples of compounds of the present invention which are labeled with aradioisotope appropriate to various diagnostic uses are for example,¹¹C— or ¹⁸F-labeled analogs of compound 14 which would be suitable foruse in positron emission tomography.

Unless otherwise stated, structures depicted herein are also meant toinclude compounds which differ only in the presence of one or moreisotopically enriched atoms. For example, compounds having the presentstructure except for the replacement of a hydrogen atom by deuterium ortritium, or the replacement of a carbon atom by ¹³C or ¹⁴C, or thereplacement of a nitrogen atom by ¹⁵N, or the replacement of an oxygenatom with ¹⁷O or ¹⁸O are within the scope of the invention. Suchisotopically labeled compounds are useful as research or diagnostictools.

In all of the examples described below, protecting groups for sensitiveor reactive groups are employed where necessary in accordance withgeneral principles of synthetic chemistry. Protecting groups aremanipulated according to standard methods of organic synthesis (T. W.Green and P. G. M. Wuts, Protecting Groups in Organic Synthesis, 3^(rd)Edition, John Wiley & Sons, New York (1999)). These groups are removedat a convenient stage of the compound synthesis using methods that arereadily apparent to those skilled in the art. The selection of processesas well as the reaction conditions and order of their execution shall beconsistent with the preparation of compounds of the present invention.

The present invention also provides a method for the synthesis of novelcompounds useful as intermediates, such as diethyl(R)-2-(1-(tert-butoxycarbonyl)pyrrolidin-3-yl)malonate (compound 3),(R)-2-(1-(tert-butoxycarbonyl)pyrrolidin-3-yl)malonic acid (compound 4),tert-butyl (R)-3-(2-hydroxyethyl)pyrrolidine-1-carboxylate (compound 6),and tert-butyl (R)-3-(2-iodoethyl)pyrrolidine-1-carboxylate (compound8).

Salt Forms

One aspect of the present invention relates to novel salt forms of(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridine.(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridinein the free base form is a viscous oil with limited water solubility.However, the free base may react with both inorganic and organic acidsto make acid addition salts that have physical properties that areadvantageous for the preparation of pharmaceutical compositions such ascrystallinity, water solubility, and stability toward chemicaldegradation.

The present invention relates to pharmaceutically acceptable salts of(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridine.Examples of suitable pharmaceutically acceptable salts include inorganicacid addition salts such as chloride, bromide, sulfate, phosphate, andnitrate; organic acid addition salts such as acetate, galactarate,propionate, succinate, lactate, glycolate, malate, tartrate, citrate,maleate, fumarate, methanesulfonate, p-toluenesulfonate, and ascorbate;salts with acidic amino acid such as aspartate and glutamate; alkalimetal salts such as sodium salt and potassium salt; alkaline earth metalsalts such as magnesium salt and calcium salt; ammonium salt; organicbasic salts such as trimethylamine salt, triethylamine salt, pyridinesalt, picoline salt, dicyclohexylamine salt, andN,N′-dibenzylethylenediamine salt; and salts with basic amino acid suchas lysine salt and arginine salt. The salts may be in some caseshydrates or solvates, such as ethanol solvates.

One embodiment of the present invention relates to acid addition saltsof(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridinewherein the acid is selected from hydrochloric acid, methane sulphonicacid, maleic acid, phosphoric acid, 1-hydroxy-2-naphthoic acid, malonicacid, L-tartaric acid, fumaric acid, citric acid, L-malic acid,R-mandelic acid, S-mandelic acid, succinic acid, 4-acetamidobenzoicacid, adipic acid, galactaric acid, di-p-toluoyl-D-tartaric acid, oxalicacid, D-glucuronic acid, 4-hydroxybenzoic acid, 4-methoxybenzoic acid,(1S)-(+)-10-camphorsulfonic acid, (1R,3S)-(+)-camphoric acid, andp-toluenesulfonic acid. The present invention also includes hydrates andsolvates of these salt forms.

The stoichiometry of the salts comprised in the present invention mayvary. For example, it is typical that the molar ratio of acid to(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineis 1:2 or 1:1, but other ratios, such as 3:1, 1:3, 2:3, 3:2 and 2:1, maybe possible and are likewise included in the scope of the presentinvention

Depending upon the manner by which the salts described herein areformed, the salts may have crystalline structures that occlude solventsthat are present during salt formation. Thus, the salts may occur ashydrates and other solvates of varying stoichiometry of solvent relativeto(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridine.

In one embodiment of the present invention, the salt has a stoichiometryof acid to(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineof 1:2. In another embodiment, the salt has a stoichiometry of acid to(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineof 1:1.

Another embodiment of the present invention relates to(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridinemono-L-malate or a hydrate or solvate thereof.

Another embodiment of the present invention relates to(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridinehemi-galactarate or a hydrate or solvate thereof.

Another embodiment of the present invention relates to(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineoxalate or a hydrate or solvate thereof.

Another embodiment of the present invention relates to(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridinedi-p-toluoyl-D-tartrate or a hydrate or solvate thereof.

One embodiment of the present invention relates to the following saltsof(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridine;

-   -   4-Acetamidobenzoic    -   Adipic    -   (1R,3S)-(+)-Camphoric    -   (1S)-(+)-1 O-Camphorsulfonic    -   Citric    -   Fumaric    -   D-glucuronic    -   Hydrochloric    -   4-Hydroxybenzoic    -   1-Hydroxy-2-naphthoic (Xinafoic)    -   Maleic    -   L-Malic    -   Malonic    -   (R)-Mandelic    -   (S)-Mandelic    -   Methanesulfonic    -   4-Methoxybenzoic    -   Phosphoric    -   Succinic    -   L-Tartaric    -   p-Toluenesulfonic.H20

or a hydrate or solvate thereof.

A further aspect of the present invention relates to processes for thepreparation of the salts. The salts may be obtained by crystallizationunder controlled conditions.

The invention also relates to a process for the preparation(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridinesalt forms comprising the following steps:

(i) mixing the free base, or a solution of the free base ofsubstantially pure(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridinein a suitable solvent, with any of the acids mentioned above in pureform or as a solution of any of the acids in a suitable solvent,typically 0.5 to 1 equivalents of the acid;(ii)(a) cooling the resulting salt solution if necessary to causeprecipitation, or(ii)(b) adding a suitable anti-solvent to cause precipitation, or(ii)(c) evaporating the solvent and adding and new solvent and repeatingeither steps (ii)(a) or step (ii)(b); and(iii) filtering and collecting the salt.

The stoichiometry, solvent mix, solute concentration, and temperatureemployed may vary.

Representative solvents that may be used to prepare or recrystallize thesalt forms include, without limitation, ethanol, methanol, propanol,2-propanol, isopropyl acetate, acetone, ethyl acetate, toluene, water,methyl ethyl ketone, methyl isobutyl ketone, tert-butyl methyl ether,tetrahydrofuran, dichloromethane, n-heptane, and acetonitrile.

In one embodiment the solvent is selected from ethanol, propanol,isopropyl acetate, water, hexane, or mixtures thereof, and thetemperature used for precipitation is between 16° C. and 25° C.

In one embodiment the acid is L-malic acid, and the solvent used is2-propoanol alone or in combination with isopropyl acetate. In anotherembodiment the acid is oxalic acid, and the solvent used is aqueous2-propanol.

In a further embodiment, the salts are(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridinemono-L-malate or hemi-galactarate or oxalate or di-p-toluoyl-D-tartrate.

The stability of the obtained salts may be demonstrated in a variety ofways.

Propensity to gain and release atmospheric moisture may be assessed bydynamic vapor sorption (DVS).

Methods of Treatment

(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridine,or pharmaceutically acceptable salts thereof, or a pharmaceuticalcomposition comprising said compounds may be used for the prevention ortreatment of various conditions or disorders for which other types ofnicotinic compounds have been proposed or are shown to be useful astherapeutics, such as CNS disorders, inflammation, inflammatory responseassociated with bacterial and/or viral infection, pain, metabolicsyndrome, autoimmune disorders or other disorders described in furtherdetail herein. The compounds may also be used as a diagnostic agent inreceptor binding studies (in vitro and in vivo). Such therapeutic andother teachings are described, for example, in references previouslylisted herein, including Williams et al., Drug News Perspec. 7(4): 205(1994), Arneric et al., CNS Drug Rev. 1(1): 1-26 (1995), Arneric et al.,Exp. Opin. Invest. Drugs 5(1): 79-100 (1996), Bencherif et al., J.Pharmacol. Exp. Ther. 279: 1413 (1996), Lippiello et al., J. Pharmacol.Exp. Ther. 279: 1422 (1996), Damaj et al., J. Pharmacol. Exp. Ther. 291:390 (1999); Chiari et al., Anesthesiology 91: 1447 (1999), Lavand'hommeand Eisenbach, Anesthesiology 91: 1455 (1999), Holladay et al., J. Med.Chem. 40(28): 4169-94 (1997), Bannon et al., Science 279: 77 (1998), PCTWO 94/08992, PCT WO 96/31475, PCT WO 96/40682, and U.S. Pat. No.5,583,140 to Bencherif et al., U.S. Pat. No. 5,597,919 to Dull et al.,U.S. Pat. No. 5,604,231 to Smith et al. and U.S. Pat. No. 5,852,041 toCosford et al.

One embodiment of the present invention relates to use of(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineor a pharmaceutically acceptable salt thereof in the manufacture of amedicament.

Another embodiment of the present invention relates to use of(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridinemono-L-malate or hemi-galactarate or oxalate or di-p-toluoyl-D-tartratefor use as a medicament.

One embodiment of the present invention relates to a method for thetreatment or prevention of central nervous system (CNS) disorders,comprising administering to a mammal in need of such treatment, atherapeutically effective amount of(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineor a pharmaceutically acceptable salt thereof or the mono-L-malate orhemi-galactarate or oxalate or di-p-toluoyl-D-tartrate. Morespecifically, the disorder may be selected from the group consisting ofCNS disorders, inflammation, inflammatory response associated withbacterial and/or viral infection, pain, metabolic syndrome, autoimmunedisorders or other disorders described in further detail herein.

One embodiment of the present invention relates to a pharmaceuticalcomposition comprising a therapeutically effective amount of(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineor a pharmaceutically acceptable salt thereof or the mono-L-malate orhemi-galactarate or oxalate or di-p-toluoyl-D-tartrate salt thereof andone or more pharmaceutically acceptable carrier, diluents, excipients,or adjuvant.

One embodiment of the present invention relates to the use of apharmaceutical composition of the present invention in the manufactureof a medicament for treatment of CNS disorders.

Another embodiment of the present invention relates to use of(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineor a pharmaceutically acceptable salt thereof, or the mono-L-malate orhemi-galactarate or oxalate or di-p-toluoyl-D-tartrate salt thereof, inthe manufacture of a medicament for treatment or prevention of disordersmediated by NNR.

Another embodiment of the present invention relates to a method ofmodulating NNR in a subject in need thereof through the administrationof(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineor a pharmaceutically acceptable salt thereof or the mono-L-malate orhemi-galactarate or oxalate or di-p-toluoyl-D-tartrate salt thereof.

CNS Disorders

((R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridine,or a pharmaceutically acceptable salt thereof, or the mono-L-malate orhemi-galactarate or oxalate or di-p-toluoyl-D-tartrate salt thereof, ora pharmaceutical composition comprising said compounds are useful in thetreatment or prevention of a variety of CNS disorders, includingneurodegenerative disorders, neuropsychiatric disorders, neurologicdisorders, and addictions. The compounds and their pharmaceuticalcompositions may be used to treat or prevent cognitive deficits anddysfunctions, age-related and otherwise; attentional disorders anddementias, including those due to infectious agents or metabolicdisturbances; to provide neuroprotection; to treat convulsions andmultiple cerebral infarcts; to treat mood disorders, compulsions andaddictive behaviors; to provide analgesia; to control inflammation, suchas mediated by cytokines and nuclear factor kappa B; to treatinflammatory disorders; to provide pain relief; and to treat infections,as anti-infectious agents for treating bacterial, fungal, and viralinfections. Among the disorders, diseases and conditions that thecompounds and pharmaceutical compositions of the present invention maybe used to treat or prevent are: age-associated memory impairment(AAMI), mild cognitive impairment (MCI), age-related cognitive decline(ARCD), pre-senile dementia, early onset Alzheimer's disease, seniledementia, dementia of the Alzheimer's type, Alzheimer's disease,cognitive impairment no dementia (CIND), Lewy body dementia,HIV-dementia, AIDS dementia complex, vascular dementia, Down syndrome,head trauma, traumatic brain injury (TBI), dementia pugilistica,Creutzfeld-Jacob Disease and prion diseases, stroke, ischemia, attentiondeficit disorder, attention deficit hyperactivity disorder, dyslexia,schizophrenia, schizophreniform disorder, schizoaffective disorder,cognitive dysfunction in schizophrenia, cognitive deficits inschizophrenia, Parkinsonism including Parkinson's disease,postencephalitic parkinsonism, parkinsonism-dementia of Gaum,frontotemporal dementia Parkinson's Type (FTDP), Pick's disease,Niemann-Pick's Disease, Huntington's Disease, Huntington's chorea,tardive dyskinesia, hyperkinesia, progressive supranuclear palsy,progressive supranuclear paresis, restless leg syndrome,Creutzfeld-Jakob disease, multiple sclerosis, amyotrophic lateralsclerosis (ALS), motor neuron diseases (MND), multiple system atrophy(MSA), corticobasal degeneration, Guillain-Barré Syndrome (GBS), andchronic inflammatory demyelinating polyneuropathy (CIDP), epilepsy,autosomal dominant nocturnal frontal lobe epilepsy, mania, anxiety,depression, premenstrual dysphoria, panic disorders, bulimia, anorexia,narcolepsy, excessive daytime sleepiness, bipolar disorders, generalizedanxiety disorder, obsessive compulsive disorder, rage outbursts,oppositional defiant disorder, Tourette's syndrome, autism, drug andalcohol addiction, tobacco addiction, and eating disorders.

Cognitive impairments or dysfunctions may be associated with psychiatricdisorders or conditions, such as schizophrenia and other psychoticdisorders, including but not limited to psychotic disorder,schizophreniform disorder, schizoaffective disorder, delusionaldisorder, brief psychotic disorder, shared psychotic disorder, andpsychotic disorders due to a general medical conditions, dementias andother cognitive disorders, including but not limited to mild cognitiveimpairment, pre-senile dementia, Alzheimer's disease, senile dementia,dementia of the Alzheimer's type, age-related memory impairment, Lewybody dementia, vascular dementia, AIDS dementia complex, dyslexia,Parkinsonism including Parkinson's disease, cognitive impairment anddementia of Parkinson's Disease, cognitive impairment of multiplesclerosis, cognitive impairment caused by traumatic brain injury,dementias due to other general medical conditions, anxiety disorders,including but not limited to panic disorder without agoraphobia, panicdisorder with agoraphobia, agoraphobia without history of panicdisorder, specific phobia, social phobia, obsessive-compulsive disorder,post-traumatic stress disorder, acute stress disorder, generalizedanxiety disorder and generalized anxiety disorder due to a generalmedical condition, mood disorders, including but not limited to majordepressive disorder, dysthymic disorder, bipolar depression, bipolarmania, bipolar I disorder, depression associated with manic, depressiveor mixed episodes, bipolar II disorder, cyclothymic disorder, and mooddisorders due to general medical conditions, sleep disorders, includingbut not limited to dyssomnia disorders, primary insomnia, primaryhypersomnia, narcolepsy, parasomnia disorders, nightmare disorder, sleepterror disorder and sleepwalking disorder, mental retardation, learningdisorders, motor skills disorders, communication disorders, pervasivedevelopmental disorders, attention-deficit and disruptive behaviordisorders, attention deficit disorder, attention deficit hyperactivitydisorder, feeding and eating disorders of infancy, childhood, or adults,tic disorders, elimination disorders, substance-related disorders,including but not limited to substance dependence, substance abuse,substance intoxication, substance withdrawal, alcohol-related disorders,amphetamine or amphetamine-like-related disorders, caffeine-relateddisorders, cannabis-related disorders, cocaine-related disorders,hallucinogen-related disorders, inhalant-related disorders,nicotine-related disorders, opioid-related disorders, phencyclidine orphencyclidine-like-related disorders, and sedative-, hypnotic- oranxiolytic-related disorders, personality disorders, including but notlimited to obsessive-compulsive personality disorder and impulse-controldisorders.

The above conditions and disorders are discussed in further detail, forexample, in the American Psychiatric Association: Diagnostic andStatistical Manual of Mental Disorders, Fourth Edition, Text Revision,Washington, D.C., American Psychiatric Association, 2000. This Manualmay also be referred to for greater detail on the symptoms anddiagnostic features associated with substance use, abuse, anddependence.

One embodiment relates to a method of treating or preventing CNSdisorders in a subject in need thereof comprising administering to saidsubject((R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridine,or a pharmaceutically acceptable salt thereof, or the mono-L-malate orhemi-galactarate or oxalate or di-p-toluoyl-D-tartrate salt thereof, ora pharmaceutical composition comprising said compounds.

In another embodiment the CNS disorders are selected from cognitivedysfunction in schizophrenia (CDS), Alzheimers Disease (AD), attentiondeficit disorder (ADD), pre-senile dementia (early onset of Alzheimer'sDisease), dementia of the Alzheimer's type, mild cognitive impairment,age associated memory impairment and attention deficit hyperactivitydisorder (ADHD). In one embodiment the CNS disorders are selected frommemory improvement and learning improvement.

Inflammation

The nervous system, primarily through the vagus nerve, is known toregulate the magnitude of the innate immune response by inhibiting therelease of macrophage tumor necrosis factor (TNF). This physiologicalmechanism is known as the “cholinergic anti-inflammatory pathway” (see,for example, Tracey, “The inflammatory reflex,” Nature 420: 853-9(2002)). Excessive inflammation and tumor necrosis factor synthesiscause morbidity and even mortality in a variety of diseases. Thesediseases include, but are not limited to, endotoxemia, rheumatoidarthritis, osteoarthritis, psoriasis, asthma, atherosclerosis,idiopathic pulmonary fibrosis, and inflammatory bowel disease.

Inflammatory conditions that may be treated or prevented byadministering the compounds described herein include, but are notlimited to, chronic and acute inflammation, psoriasis, endotoxemia,gout, acute pseudogout, acute gouty arthritis, arthritis, rheumatoidarthritis, osteoarthritis, allograft rejection, chronic transplantrejection, asthma, atherosclerosis, mononuclear-phagocyte dependent lunginjury, idiopathic pulmonary fibrosis, atopic dermatitis, chronicobstructive pulmonary disease, adult respiratory distress syndrome,acute chest syndrome in sickle cell disease, inflammatory bowel disease,Crohn's disease, ulcerative colitis, acute cholangitis, aphteousstomatitis, pouchitis, glomerulonephritis, lupus nephritis, thrombosis,and graft vs. host reaction.

Inflammatory Response Associated with Bacterial and/or Viral Infection

Many bacterial and/or viral infections are associated with side effectsbrought on by the formation of toxins, and the body's natural responseto the bacteria or virus and/or the toxins. As discussed above, thebody's response to infection often involves generating a significantamount of TNF and/or other cytokines. The over-expression of thesecytokines can result in significant injury, such as septic shock (whenthe bacteria is sepsis), endotoxic shock, urosepsis, viral pneumonitis,and toxic shock syndrome.

Cytokine expression is mediated by NNRs, and may be inhibited byadministering agonists or partial agonists of these receptors. Thosecompounds described herein that are agonists or partial agonists ofthese receptors may therefore be used to minimize the inflammatoryresponse associated with bacterial infection, as well as viral andfungal infections. Examples of such bacterial infections includeanthrax, botulism, and sepsis. Some of these compounds may also haveantimicrobial properties.

The compounds of the present invention may also be used as adjuncttherapy in combination with existing therapies to manage bacterial,viral and fungal infections, such as antibiotics, antivirals andantifungals. Antitoxins may also be used to bind to toxins produced bythe infectious agents and allow the bound toxins to pass through thebody without generating an inflammatory response. Examples of antitoxinsare disclosed, for example, in U.S. Pat. No. 6,310,043 to Bundle et al.Other agents effective against bacterial and other toxins may beeffective and their therapeutic effect may be complemented byco-administration with the compounds described herein.

Pain

The compounds may be administered to treat and/or prevent pain,including acute, neurologic, inflammatory, neuropathic and chronic pain.The analgesic activity of compounds described herein may be demonstratedin models of persistent inflammatory pain and of neuropathic pain,performed as described in U.S. Published Patent Application No.20010056084 A1 (Allgeier et al.) (e.g., mechanical hyperalgesia in thecomplete Freund's adjuvant rat model of inflammatory pain and mechanicalhyperalgesia in the mouse partial sciatic nerve ligation model ofneuropathic pain).

The analgesic effect is suitable for treating pain of various genesis oretiology, in particular in treating inflammatory pain and associatedhyperalgesia, neuropathic pain and associated hyperalgesia, chronic pain(e.g., severe chronic pain, post-operative pain and pain associated withvarious conditions including cancer, angina, renal or biliary colic,menstruation, migraine and gout). Inflammatory pain may be of diversegenesis, including arthritis and rheumatoid disease, teno-synovitis andvasculitis. Neuropathic pain includes trigeminal or herpetic neuralgia,neuropathies, diabetic neuropathy pain, causalgia, low back pain anddeafferentation syndromes such as brachial plexus avulsion.

One embodiment relates to a method of treating pain in a subject in needthereof comprising administering to said subject((R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridine,or a pharmaceutically acceptable salt thereof, or the mono-L-malate orhemi-galactarate or oxalate or di-p-toluoyl-D-tartrate salt thereof, ora pharmaceutical composition comprising said compounds.

Other Disorders

In addition to treating CNS disorders, inflammation, and pain, thecompounds of the present invention may be also used to prevent or treatcertain other conditions, diseases, and disorders in which NNRs play arole. Examples include autoimmune disorders such as lupus, disordersassociated with cytokine release, cachexia secondary to infection (e.g.,as occurs in AIDS, AIDS related complex and neoplasia), obesity,pemphitis, urinary incontinence, retinal diseases, infectious diseases,myasthenia, Eaton-Lambert syndrome, dystonia, hypertension,osteoporosis, vasoconstriction, vasodilatation, cardiac arrhythmias,type I diabetes, type II diabetes, ulcers, bulimia, anorexia,constipation, and diarrhea, as well as those indications set forth inpublished PCT application WO 98/25619. The compounds of this inventionmay also be administered to treat convulsions such as those that aresymptomatic of epilepsy, and to treat conditions such as syphillis andCreutzfeld-Jakob disease.

Diagnostic Uses

Another embodiment of the present invention relates to compounds thathave utility as diagnostic agents and in receptor binding studies asdescribed herein.

The compounds may be used in diagnostic compositions, such as probes,particularly when they are modified to include appropriate labels. Theprobes may be used, for example, to determine the relative number and/orfunction of specific receptors, particularly the α4β2 receptor subtype.For this purpose the compounds of the present invention most preferablyare labeled with a radioactive isotopic moiety such as ¹¹C, ¹⁸F, ⁷⁶Br,¹²³I or ¹²³I.

One embodiment of the invention relates to((R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridine,or a pharmaceutically acceptable salt thereof, or the mono-L-malate orhemi-galactarate or oxalate or di-p-toluoyl-D-tartrate salt thereof,wherein one to three of the atoms represents a detectable isotopeselected from ³H, ¹⁹F and ¹³C, or wherein one of the atoms is adetectable isotope selected from ¹⁸F, ¹¹C and ¹⁴C.

The administered compounds may be detected using known detection methodsappropriate for the label used. Examples of detection methods arescintillation counting, position emission topography (PET),single-photon emission computed tomography (SPECT), gamma imaging,magnetic resonance imaging (MRI) or magnetic resonance spectroscopy(MRS). The radiolabels described above are useful in PET (e.g., ¹¹C, ¹⁸For ⁷⁶Br) and SPECT (e.g., ¹²³I) imaging, with half-lives of about 20.4min for ¹¹C, about 109 min for ¹⁸F, about 13 h for ¹²³I, and about 16 hfor ⁷⁶Br. A high specific activity is desired to visualize the selectedreceptor subtypes at non-saturating concentrations. The administereddoses typically are low and provide high contrast images. Determinationof dose is carried out in a manner known to one skilled in the art ofradiolabel imaging. The compounds may be administered in compositionsthat incorporate other ingredients, such as those types of ingredientsthat are useful in formulating a diagnostic composition. Compoundsuseful in accordance with carrying out the present invention mostpreferably are employed in forms of high purity. After the compounds areadministered to a subject (e.g., a human subject), the presence of thatcompound within the subject may be imaged and quantified by appropriatetechniques in order to indicate the presence, quantity, andfunctionality of selected NNR subtypes. In addition to humans, thecompounds may also be administered to animals, such as mice, rats,horses, dogs, and monkeys. SPECT and PET imaging may be carried outusing any appropriate technique and apparatus. The radiolabeledcompounds bind with high affinity to selective NNR subtypes (e.g., α4β2)and preferably exhibit negligible non-specific binding to othernicotinic cholinergic receptor subtypes (e.g., those receptor subtypesassociated with muscle and ganglia). As such, the compounds may be usedas agents for noninvasive imaging of nicotinic cholinergic receptorsubtypes within the body of a subject, particularly within the brain fordiagnosis associated with a variety of CNS diseases and disorders.

In one aspect, the diagnostic compositions may be used in a method todiagnose disease in a subject, such as a human patient. The methodinvolves administering to that patient a detectably labeled compound asdescribed herein, and detecting the binding of that compound to selectedNNR subtypes (e.g., α4β2 receptor subtypes). Those skilled in the art ofusing diagnostic tools, such as PET and SPECT, can use the radiolabeledcompounds described herein to diagnose a wide variety of conditions anddisorders, including conditions and disorders associated withdysfunction of the central and autonomic nervous systems. Such disordersinclude a wide variety of CNS diseases and disorders, such asAlzheimer's disease, Parkinson's disease, and schizophrenia or anydisorder herein mentioned.

Receptor Binding

The compounds of this invention may be used as reference ligands inbinding assays for compounds which bind to NNR subtypes, particularlythe α4β2 receptor subtypes. For this purpose the compounds of thisinvention are preferably labeled with a radioactive isotopic moiety suchas ³H, or ¹⁴C. Examples of such binding assays are described in detailbelow.

Pharmaceutical Compositions

In one aspect the present invention relates to pharmaceuticalcompositions comprising the compound of the present invention and one ormore pharmaceutically acceptable carrier, diluent, or excipient. Anotheraspect of the invention provides a process for the preparation of apharmaceutical composition including admixing the compound of thepresent invention with one or more pharmaceutically acceptable carrier,diluent, or excipient.

The manner in which the compound of the present invention isadministered may vary. The compound of the present invention ispreferably administered orally. Preferred pharmaceutical compositionsfor oral administration include tablets, capsules, caplets, syrups,solutions, and suspensions. The pharmaceutical compositions of thepresent invention may be provided in modified release dosage forms suchas time-release tablet and capsule formulations.

The pharmaceutical compositions may also be administered via injection,namely, intravenously, intramuscularly, subcutaneously,intraperitoneally, intraarterially, intrathecally, andintracerebroventricularly. Carriers for injection may include 5%dextrose solutions, saline, and phosphate buffered saline.

The compositions may also be administered using other means, forexample, rectal administration. The compounds may also be administeredby inhalation, for example, in the form of an aerosol; topically, suchas, in lotion form; transdermally, such as, using a transdermal patch(for example, by using technology that is commercially available fromNovartis and Alza Corporation), by powder injection, or by buccal,sublingual, or intranasal absorption.

Pharmaceutical compositions may be formulated in unit dose form, or inmultiple or subunit doses forms.

The administration of the pharmaceutical compositions described hereinmay be intermittent, or at a gradual, continuous, constant or controlledrate. The pharmaceutical compositions may be administered to awarm-blooded animal, for example, a mammal such as a mouse, rat, cat,guinea pig, rabbit, horses, dog, pig, cow, or monkey; but advantageouslyis administered to a human being.

Combinations

The compound of the present invention may be used in the treatment of avariety of disorders and conditions and, as such, may be used incombination with a variety of other therapeutic agents useful in thetreatment or prophylaxis of those disorders. Thus, one embodiment of thepresent invention relates to the administration of the compound of thepresent invention in combination with other therapeutic agents. Forexample, the compound of the present invention may be used incombination with other NNR ligands (such as varenicline), antioxidants(such as free radical scavenging agents), antibacterial agents (such aspenicillin antibiotics), antiviral agents (such as nucleoside analogs,like zidovudine and acyclovir), anticoagulants (such as warfarin),anti-inflammatory agents (such as NSAIDs), anti-pyretics, analgesics,anesthetics (such as used in surgery), acetylcholinesterase inhibitors(such as donepezil and galantamine), antipsychotics (such ashaloperidol, clozapine, olanzapine, and quetiapine), immuno-suppressants(such as cyclosporin and methotrexate), neuroprotective agents, steroids(such as steroid hormones), corticosteroids (such as dexamethasone,predisone, and hydrocortisone), vitamins, minerals, nutraceuticals,anti-depressants (such as imipramine, fluoxetine, paroxetine,escitalopram, sertraline, venlafaxine, and duloxetine), anxiolytics(such as alprazolam and buspirone), anticonvulsants (such as phenyloinand gabapentin), vasodilators (such as prazosin and sildenafil), moodstabilizers (such as valproate and aripiprazole), anti-cancer drugs(such as anti-proliferatives), antihypertensive agents (such asatenolol, clonidine, amlopidine, verapamil, and olmesartan), laxatives,stool softeners, diuretics (such as furosemide), anti-spasmotics (suchas dicyclomine), anti-dyskinetic agents, and anti-ulcer medications(such as esomeprazole). Such a combination of therapeutic agents may beadministered together or separately and, when administered separately,administration may occur simultaneously or sequentially, in any order.The amounts of the compounds or agents and the relative timings ofadministration will be selected in order to achieve the desiredtherapeutic effect. The administration in combination of a compound ofthe present invention with other therapeutic agents may be incombination by administration concomitantly in: (1) a unitarypharmaceutical composition including both compounds; or (2) separatepharmaceutical compositions each including one of the compounds.Alternatively, the combination may be administered separately in asequential manner wherein one treatment agent is administered first andthe other second. Such sequential administration may be close in time orremote in time.

Another aspect of the present invention relates to combination therapycomprising administering to the subject a therapeutically orprophylactically effective amount of the compound of the presentinvention and one or more other therapeutic agents includingchemotherapeutics, radiation therapuetic agents, gene therapeuticagents, or agents used in immunotherapy.

Low Dose

((R)-3-((E)-2-(Pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineor a salt thereof is a substrate for Pgp brain pump which is located inthe blood brain barrier. The Pgp pump is responsible for pumpingsubstances out of the brain. Due to this pump, it is often difficult toget drugs into the brain in therapeutically effective amounts. Thisoften results in the administration of high doses of drugs, which atthese high levels may have side effects in other parts of the humanbody.

((R)-3-((E)-2-(Pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridine,although being a substrate for the PGP pump, can be administered at lowdoses while at the same time have a relatively long duration of effect.For example, compared to acetylcholine, the natural agonist for NNR, theresponse for((R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineis twice as great in the in vitro assay at α4β2.

One embodiment of the invention relates to administration of apharmaceutical composition comprising((R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridine,or a pharmaceutically acceptable salt thereof, in certain embodiments,the mono-L-malate or hemi-galactarate or oxalate ordi-p-toluoyl-D-tartrate salt thereof, in amounts of between 1 to 2200μg/day. In another embodiment the amount is 50 to 1500 μg/day. In afurther embodiment the amount is 50 to 1000 μg/day. In one embodimentthe amount is 50 to 500 μg/day. In another embodiment the amount is 75to 300 μg/day. In yet another embodiment the amount is 75 to 200 μg/day.In yet a further embodiment the amount is 75 to 150 μg/day.

The dose of((R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridine,or a pharmaceutically acceptable salt thereof, in certain embodiments,the mono-L-malate or hemi-galactarate or oxalate ordi-p-toluoyl-D-tartrate salt thereof, may be administered one, two, orthree times daily. One embodiment relates to once daily administration.Another embodiment relates to twice daily administration.

Another embodiment of the invention relates to a NNR agonist which has ahalf life (t_(1/2)) between 5 and 8 hours. In one embodiment the t_(1/2)is between 6 and 7 hours. In another embodiment the t_(1/2) is 6.8hours.

Another embodiment of the invention relates to a NNR agonist which has aduration of action between 5 and 10 hours. In one embodiment theduration is between 6 to 9 hours.

In a further embodiment the duration is 8 hours.

In a further embodiment the agonist is an α4β2 agonist.

In yet another embodiment the agonist is((R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridine.

EXAMPLES

The following examples are provided to illustrate the present invention,and should not be construed as limiting thereof. In these examples, allparts and percentages are by weight, unless otherwise noted.

Example 1 Instrumentation and Experimental Protocols forCharacterization of(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineand its Salt Forms Nuclear Magnetic Resonance (NMR) Spectrometry

NMR spectra were collected on either a Varian Unity 300 MHz instrumentor a Bruker 400 MHz instrument equipped with an auto-sampler andcontrolled by a DRX400 console. Automated experiments were acquiredusing ICONNMR v4.0.4 (build 1) running with Topspin v 1.3 (patch level8) using the standard Bruker loaded experiments. For non-routinespectroscopy, data were acquired through the use of Topspin alone.

Melting Point

A Fisher-Johns hot stage melting point apparatus was used, at a settingcorresponding to a heating rate of about 5° C. per min.

Differential Scanning Calorimetry (DSC)

DSC data were collected on a TA Instruments Q1000 or a Mettler DSC 823eequipped with a 50 position auto-sampler. The instrument was calibratedfor energy and temperature calibration using certified indium. Typically0.5-1.5 mg of each sample, in a pin-holed aluminium pan, was heated at10° C./min from 25° C. to 175-200° C. A nitrogen purge at 30 mL/min wasmaintained over the sample.

Dynamic Vapor Sorption (DVS)

Sorption isotherms were determined using a SMS DVS Intrinsic moisturesorption analyzer controlled by SMS Analysis suite software. The sampletemperature was maintained at 25° C. by the instrument controls. Thehumidity was controlled by mixing streams of dry and wet nitrogen, witha total flow rate of 200 mL/min. The relative humidity was measured by acalibrated Rotronic probe (dynamic range of 1.0-100% RH), located nearthe sample. The weight change, (mass relaxation) of the sample as afunction of % RH was constantly monitored by the microbalance (accuracy±0.005 mg).

Typically a 5-20 mg sample was placed on the tared mesh stainless steelbasket under ambient conditions. The sample was loaded and unloaded at40% RH and 25° C. (typical ambient conditions). A moisture sorptionisotherm was performed as outlined below (2 scans giving 1 completecycle). The standard isotherm was performed at 25° C. at 10% RHintervals over a 0-90% RH range.

DVS Generic Method Parameters

Parameters Values Adsorption - Scan 1 40-90 Desorption/Adsorption - Scan2 90-Dry, Dry-40 Intervals (% RH) 10 Number of Scans 2 Flow rate(mL/min) 200 Temperature (° C.) 25 Stability (° C./min) 0.2 SorptionTime (h) 6 hour time out

Chemical Purity by HPLC

Purity analysis was performed on an Agilent HP1100 series systemequipped with a diode array detector and using ChemStation softwarevB.02.01-SR1. HPLC method parameters for chemical purity determination:

Sample Preparation 0.5 mg/mL in acetonitrile:water 1:1 (v/v) Column:Phenomenex Luna C18 (2), 150 × 4.6 mm, 5 μm Column Temperature 25 (°C.): Injection (μL): 5 Detection: 255, 90 Wavelength, Bandwidth(nm):Flow Rate (mL/min): 1 Phase A: 0.1% TFA in water Phase B: 0.085% TFA inacetonitrile Timetable: Time (min) % Phase A % Phase B 0 95 5 25 5 9525.2 95 5 30 95 5

Ion Chromatography

Data were collected on a Metrohm 761 Advanced Compact IC (for cations)and a Metrohm 861 Advanced Compact IC (for anions) using IC Net softwarev2.3. Samples were prepared as 1000 ppm stocks in DMSO. Samples werediluted to 100 ppm with DMSO prior to testing. Quantification wasachieved by comparison with standard solutions of known concentration ofthe ion being analyzed.

Ion Chromatography Method for Anions:

Type of method Anion exchange Column: Metrosep A Supp 5 - 250 (4.0 × 250mm) Column Temperature (° C.): Ambient Injection (μL): 20 Detection:Conductivity detector Flow Rate (mL/min): 0.7 Eluent: 3.2 mM sodiumcarbonate, 1.0 mM sodium hydrogen carbonate in water

Ion Chromatography Method for Cations:

Type of method Cation exchange Column: Metrosep C 2 - 250 (4.0 × 250 mm)Column Temperature (° C.): Ambient Injection (μL): 20 Detection:Conductivity detector Flow Rate (mL/min): 1.0 Eluent: 4.0 mM Tartaricacid, 0.75 mM Dipicolinic acid in water

Example 2 Synthesis of tert-butyl(R)-3-(methylsulfonyloxy)pyrrolidine-1-carboxylate (2)

Procedure A:

To a solution of tert-butyl (R)-3-hydroxypyrrolidine-1-carboxylate (200g, 1.07 mol) and triethylamine (167 g, 1.63 mol) in toluene (700 mL) at−20 to −30° C. was added methanesulfonyl chloride (156 g, 1.36 mol)drop-wise while maintaining the temperature at −10 to −20° C. Thesolution was warmed to ambient temperature and allowed to stir. Thereaction solution was sampled hourly and analyzed by HPLC to establishcompletion of the reaction. Upon completion of the reaction, thesuspension was filtered to remove the triethylamine hydrochloride. Thefiltrate was washed with ˜600 mL of dilute aqueous sodium bicarbonatesolution. The organic layer was dried and concentrated under reducedpressure to give 2 as a viscous oil (260 g, 92%) which is used withoutfurther purification. ¹H NMR (CDCl₃, 400 MHz) δ 5.27 (m, 1H), 3.44-3.76(m, 4H), 3.05 (s, 3H), 2.26 (m, 1H), 2.15 (m, 1H), 1.47 (s, 9H).

Procedure B:

A reactor was charged with tert-butyl(R)-3-hydroxypyrrolidine-1-carboxylate (2.00 kg, 10.7 mol), toluene(8.70 kg) and triethylamine (1.75 kg, 17.3 mol). The reactor was flushedwith nitrogen for 15 min. The mixture was stirred and cooled to 3° C.Methanesulfonyl chloride (1.72 kg, mol) was slowly added (over a 2 hperiod) with continuous ice bath cooling (exothermic reaction) (aftercomplete addition, the temperature was 14° C.). The mixture, now viscouswith precipitated triethylamine hydrochloride, was stirred 12 h as itwarmed to 20° C. Both GC and TLC analysis (ninhydrin stain) indicatedthat no starting material remained. The mixture was filtered to removethe triethylamine hydrochloride, and the filtrate was returned to thereactor. The filtrate was then washed (2×3 kg) with 5% aqueous sodiumbicarbonate, using 15 min of stirring and 15 min of settling time foreach wash. The resulting organic layer was dried over anhydrous sodiumsulfate and filtered. The volatiles were removed from the filtrate undervacuum, first at 50° C. for 4 h and then at ambient temperature for 10h. The residue weighed 3.00 kg (106% yield) and was identical bychromatographic and NMR analysis to previously prepared samples, withthe exception that it contained toluene.

Example 3 Synthesis of diethyl(R)-2-(1-(tert-butoxycarbonyl)pyrrolidin-3-yl)malonate (3)

Preparation A:

To a solution of potassium tert-butoxide (187 g, 1.62 mol) in1-methyl-2-pyrrolidinone (1.19 L) was added diethyl malonate (268 g.1.67 mol) while maintaining the temperature below 35° C. The solutionwas heated to 40° C. and stirred for 20-30 min. tert-Butyl(R)-3-(methylsulfonyloxyl)pyrrolidine-1-carboxylate (112 g, 420 mmol)was added and the solution was heated to 65° C. and stirred for 6 h. Thereaction solution was sampled every 2 h and analyzed by HPLC toestablish completion of the reaction. Upon completion of reaction (10-12h), the mixture was cooled to around 25° C. De-ionized water (250 mL)was added to the solution, and the pH was adjusted to 3-4 by addition of2N hydrochloric acid (650 mL). The resulting suspension was filtered,and water (1.2 L) and chloroform (1.4 L) were added. The solution wasmixed thoroughly, and the chloroform layer was collected and evaporatedunder reduced pressure to give a yellow oil. The oil was dissolved inhexanes (2.00 L) and washed with deionized water (2×1.00 L). The organiclayer was concentrated under reduced pressure at 50-55° C. to give apale yellow oil (252 g) which ¹H NMR analysis indicates to be 49.1% of 3(123.8 g) along with 48.5% diethyl malonate (122 g), and 2% of1-methyl-2-pyrrolidinone (5 g). The material was carried forward intothe next step without further purification. ¹H NMR (CDCl₃, 400 MHz) δ4.20 (q, 4H), 3.63 (m, 1H), 3.48 (m, 1H), 3.30 (m, 1H), 3.27 (d, J=10Hz, 1H), 3.03 (m, 1H), 2.80 (m, 1H), 2.08 (m, 1H), 1.61 (m, 1H), 1.45(s, 9H), 1.27 (t, 6H).

Preparation B:

A reactor, maintained under a nitrogen atmosphere, was charged with 200proof ethanol (5.50 kg) and 21% (by weight) sodium ethoxide in ethanol(7.00 kg, 21.6 mol). The mixture was stirred and warmed to 30° C.Diethyl malonate (3.50 kg, 21.9 mol) was added over a 20 min period. Thereaction mixture was then warmed at 40° C. for 1.5 h. A solution oftert-butyl (R)-3-(methylsulfonyloxyl)pyrrolidine-1-carboxylate (3.00 kgof the product from Example 2, Procedure B, 10.7 mol) in 200 proofethanol (5.50 kg) was added, and the resulting mixture was heated atreflux (78° C.) for 2 h. Both GC and TLC analysis (ninhydrin stain)indicated that no starting material remained. The stirred mixture wasthen cooled to 25° C., diluted with water (2.25 kg), and treated slowlywith a solution of concentrated hydrochloric acid (1.27 kg, 12.9 mol) inwater (5.44 kg). This mixture was washed twice with methyl tert-butylether (MTBE) (14.1 kg and 11.4 kg), using 15 min of stirring and 15 minof settling time for each wash. The combined MTBE washes were dried overanhydrous sodium sulfate (1 kg), filtered and concentrated under vacuumat 50° C. for 6 h. The residue (red oil) weighed 4.45 kg and was 49%desired product by GC analysis (62% overall yield from tert-butyl(R)-3-hydroxypyrrolidine-1-carboxylate).

Example 4 Synthesis of(R)-2-(1-(tert-butoxycarbonyl)pyrrolidin-3-yl)malonic acid (4)

Procedure A:

To a solution of the product of Example 3, Procedure A (232 g),containing 123.8 g (380 mmol) of 3 and 121.8 g (760 mmol) of diethylmalonate, in tetrahydrofuran (1.2 L) was added a 21% potassium hydroxidesolution (450 g in 0.50 L of deionized water) while maintaining thetemperature below 25° C. The reaction mixture was heated to 45° C. andstirred for 1 h. The reaction solution was sampled every hour andanalyzed by HPLC to establish completion of the reaction. Uponcompletion of reaction (2-3 h), the mixture was cooled to around 25° C.The aqueous layer was collected and cooled to 5° C. The pH was adjustedto 2 by addition of 4N hydrochloric acid (750 mL), and the resultingsuspension was held at 5-10° C. for 30 min. The mixture was filtered,and the filter cake was washed with hexanes (1 L). The aqueous filtratewas extracted with chloroform (1 L) and the chloroform layer was putaside. The solids collected in the filtration step were re-dissolved inchloroform (1 L) by heating to 40° C. The solution was filtered toremove un-dissolved inorganic solids. The chloroform layers werecombined and concentrated under reduced pressure at 50-55° C. to give anoff-white solid (15 g). The solids were combined and dissolved in ethylacetate (350 mL) to give a suspension that was warmed to 55-60° C. for 2h. The suspension was filtered while hot and the resulting cake washedwith ethyl acetate (2×150 mL) and hexanes (2×250 mL) to give 83.0 g(80.1%) of 4 as a white solid which was used in the next step withoutfurther purification. ¹H NMR (d₄-CH₃OH, 400 MHz) δ 3.60 (m, 1H), 3.46(m, 1H), 3.29-3.32 (m, 2H), 2.72 (m, 1H), 2.09 (m, 1H), 1.70 (m, 1H),1.45 (s, 9H).

Procedure B:

A solution of the product of Example 3, Procedure B (4.35 kg),containing 2.13 kg (6.47 mol) of 3, in tetrahydrofuran (13.9 kg) wasadded to a stirred, cooled solution of potassium hydroxide (1.60 kg,40.0 mol) in deionized water (2.00 kg) under a nitrogen atmosphere,while maintaining the temperature below 35° C. The reaction mixture washeated and maintained at 40-45° C. for 24 h, by which time GC and TLCanalysis indicated that the reaction was complete. The mixture wascooled to 25° C. and washed with MTBE (34 kg), using 15 min of stirringand 15 min of settling time. The aqueous layer was collected and cooledto 1° C. A mixture of concentrated hydrochloric acid (2.61 kg, 26.5 mol)in deionized water (2.18 kg) was then added slowly, keeping thetemperature of the mixture at <15° C. during and for 15 min after theaddition. The pH of the solution was adjusted to 3.7 by further additionof hydrochloric acid. The white solid was collected by filtration,washed with water (16 kg), and vacuum dried at ambient temperature for 6d. The dry solid weighed 1.04 kg. The filtrate was cooled to <10° C. andkept at that temperature as the pH was lowered by addition of morehydrochloric acid (1.6 L of 6 N was used; 9.6 mol; final pH=2). Thewhite solid was collected by filtration, washed with water (8 L), andvacuum dried at 40° C. for 3 d. The dry solid weighed 0.25 kg. Thecombined solids (1.29 kg, 73% yield) were chromatographically identicalto previously prepared samples.

Example 5 Synthesis of(R)-2-(1-(tert-butoxycarbonyl)pyrrolidine-3-yl)acetic acid (5)

Procedure A:

A solution of (R)-2-(1-(tert-butoxycarbonyl)pyrrolidin-3-yl)malonic acid(83 g) in 1-methyl-2-pyrrolidinone (0.42 L) was stirred under nitrogenat 110-112° C. for 2 h. The reaction solution was sampled every hour andanalyzed by HPLC to establish completion of the reaction. Uponcompletion of reaction the reaction solution was cooled to 20-25° C. Thesolution was mixed with de-ionized water (1.00 L), and MTBE (1.00 L) wasadded. The phases were separated, and the organic layer was collected.The aqueous phase was extracted with MTBE (1.00 L), then chloroform(1.00 L). The organic layers were combined and concentrated underreduced pressure at 50-55° C. to give an oil. This oil was dissolved inMTBE (2.00 L) and washed twice with 0.6N hydrochloric acid (2×1.00 L).The organic layer was collected and concentrated under reduced pressureat 50-55° C. to give a semi-solid. The semi-solid was suspended in 1:4ethyl acetate/hexanes (100 mL), heated to 50° C., held for 30 min,cooled to −10° C., and filtered. The filtrate was concentrated underreduced pressure to give an oil, which was dissolved in MTBE (250 mL)and washed twice with 0.6N hydrochloric acid (2×100 mL). The organiclayer was concentrated under reduced pressure at 50-55° C. to give asemi-solid which was suspended in 1:4 ethyl acetate/hexanes (50 mL),heated to 50° C., held for 30 min, cooled to −10° C., and filtered. Thesolids were collected, suspended in hexanes (200 mL), and collected byfiltration to give 54.0 g (77.6%) of 5. ¹H NMR (CDCl₃, 400 MHz) δ 11.00(br s, 1H), 3.63 (m, 1H), 3.45 (M, 1H), 3.30 (M, 1H), 2.97 (m, 1H), 2.58(m, 1H), 2.44 (m, 2H), 2.09 (m, 1H), 1.59 (M, 1H), 1.46 (s, 9H).

Procedure B:

A solution of (R)-2-(1-(tert-butoxycarbonyl)pyrrolidin-3-yl)malonic acid(1.04 kg, 3.81 mol) in 1-methyl-2-pyrrolidinone (6.49 kg) was stirredunder nitrogen at 110° C. for 5 h, by which time TLC and HPLC analysisindicated that the reaction was complete. The reaction mixture wascooled to 25° C. (4 h) and combined with water (12.8 kg) and MTBE (9.44kg). The mixture was stirred vigorously for 20 min, and the phases wereallowed to separate (10 h). The organic phase was collected, and theaqueous phase was combined with MTBE (9.44 kg), stirred for 15 min, andallowed to settle (45 min). The organic phase was collected, and theaqueous phase was combined with MTBE (9.44 kg), stirred for 15 min, andallowed to settle (15 min). The three organic phases were combined andwashed three times with 1N hydrochloric acid (8.44 kg portions) and oncewith water (6.39 kg), using 15 min of stirring and 15 min of settlingtime for each wash. The resulting solution was dried over anhydroussodium sulfate (2.0 kg) and filtered. The filtrate was concentratedunder reduced pressure at 31° C. (2 h) to give an solid. This solid washeated under vacuum for 4 h at 39° C. for 4 h and for 16 h at 25° C.,leaving 704 g (81%) of 5 (99.7% purity by GC).

Procedure C

(streamlined synthesis of 5, using 2 as starting material): A stirredmixture of sodium ethoxide in ethanol (21 weight percent, 343 g, 1.05mol), ethanol (anhydrous, 300 mL) and diethyl malonate (168 g, 1.05 mol)was heated to 40° C. for 1.5 h. To this mixture was added a solution of(R)-tert-butyl 3-(methylsulfonyloxy)pyrrolidine-1-carboxylate (138 g,0.592 mol) in ethanol (100 mL) and the reaction mixture was heated to78° C. for 8 h. The cooled reaction mixture was diluted with water (2.0L) and acidified to pH=3 with 6M HCl (100 mL). The aqueous ethanolmixture was extracted with toluene (1.0 L), and the organic phaseconcentrated under vacuum to afford 230 g of a red oil. The red oil wasadded at 85° C. to a 22.5 weight percent aqueous potassium hydroxide(748 g, 3.01 mol). After the addition was complete, the reactiontemperature was allowed to slowly rise to 102° C. while a distillationof ethanol ensued. When the reaction temperature had reached 102° C.,and distillation had subsided, heating was continued for an additional90 min. The reaction mixture was cooled to ambient temperature andwashed with toluene (2×400 mL). To the aqueous layer was added 600 mL 6Mhydrochloric acid, while keeping the internal temperature below 20° C.This resulted in the formation of a precipitate, starting at pH of about4-5. The suspension was filtered, and the filter cake was washed with300 mL water. The solid was dried under vacuum to afford 77 g of(R)-2-(1-(tert-butoxycarbonyl)pyrrolidin-3-yl)malonic acid as anoff-white solid (54% yield with respect to (R)-tert-butyl3-(methylsulfonyloxy)pyrrolidine-1-carboxylate). ¹H NMR (DMSO-d₆, 400MHz): δ 3.47 (m, 1H); 3.32 (m, 1H); 3.24 (m, 1H); 3.16 (m, 1H); 3.92 (m,1H); 2.86 (m, 1H); 1.95 (m, 1H); 1.59 (m, 1H); 1.39 (s, 9H).

A suspension of (R)-2-(1-(tert-butoxycarbonyl)pyrrolidin-3-yl)malonicacid (15 g, 55 mmol) in toluene (150 mL) and dimethylsulfoxide (2 mL)was heated to reflux for a period of 2 h. The mixture was allowed toreach ambient and diluted with MTBE (150 mL). The organic solution waswashed with 10% aqueous citric acid (2×200 mL), and the solvent wasremoved under vacuum to afford 11.6 g of(R)-2-(1-(tert-butoxycarbonyl)-pyrrolidin-3-yl)acetic acid as anoff-white solid (92% yield). ¹H NMR (DMSO-d₆, 400 MHz): δ 12.1 (s, 1H);3.36-3.48 (m, 1H); 3.20-3.34 (m, 1H); 3.05-3.19 (m, 1H, 2.72-2.84 (m,1H); 2.30-2.42 (m, 1H), 2.22-2.30 (m, 2H); 1.85-2.00 (m, 1H); 1.38-1.54(m, 1H), 1.35 (2, 9H).

Example 6 Synthesis of tert-butyl(R)-3-(2-hydroxyethyl)pyrrolidine-1-carboxylate (6)

Procedure A:

A solution of (R)-2-(1-(tert-butoxycarbonyl)pyrrolidine-3-yl)acetic acid(49.0 g, 214 mmol) in tetrahydrofuran (THF) (200 mL) was cooled to −10°C. 250 mL (250 mmol) of a 1M borane in THF solution was added slowly tothe flask while maintaining the temperature lower than 0° C. Thesolution was warmed to ambient temperature and stirred for 1 h. Thesolution was sampled hourly and analyzed by HPLC to establish completionof the reaction. Upon completion of the reaction, the solution wascooled to 0° C., and a 10% sodium hydroxide solution (80 mL) was addeddrop-wise over a 30 minute period to control gas evolution. The solutionwas extracted with 500 mL of a 1:1 hexanes/ethyl acetate solution. Theorganic layer was washed with saturated sodium chloride solution anddried with 10 g of silica gel. The silica gel was removed by filtrationand washed with 100 mL of 1:1 hexanes/ethyl acetate. The organic layerswere combined and concentrated under vacuum to give 6 (42 g, 91.3%) as alight-orange oil that solidified upon sitting. ¹H NMR (CDCl₃, 400 MHz) δ3.67 (m, 2H), 3.38-3.62 (m, 2H), 3.25 (m, 1H), 2.90 (m, 1H), 2.25 (m,1H), 1.98-2.05 (m, 1H), 1.61-1.69 (m, 2H), 1.48-1.59 (m, 2H), 1.46 (s,9H).

Procedure B:

Borane-THF complex (3.90 kg or L of 1M in THF, mol) was added slowly toa stirred solution of(R)-2-(1-(tert-butoxycarbonyl)pyrrolidine-3-yl)acetic acid (683 g, 3.03mol) in THF (2.5 kg), kept under nitrogen gas, and using a water bath tokeep the temperature between 23 and 28° C. The addition took 1.75 h.Stirring at 25° C. was continued for 1 h, after which time GC analysisindicated complete reaction. The reaction mixture was cooled to <10° C.and maintained below 25° C. as 10% aqueous sodium hydroxide (1.22 kg)was slowly added. The addition took 40 min. The mixture was stirred 1 hat 25° C., and then combined with 1:1 (v/v) heptane/ethyl acetate (7 L).The mixture was stirred for 15 min and allowed to separate into phases(1 h). The organic phase was withdrawn, and the aqueous phase wascombined with a second 7 L portion of 1:1 heptane/ethyl acetate. Thiswas stirred for 15 min and allowed to separate into phases (20 min). Theorganic phase was again withdrawn, and the combined organic phases werewashed with saturate aqueous sodium chloride (4.16 kg), using 15 min ofmixing and 1 h of settling time. The organic phase was combined withsilica gel (140 g) and stirred 1 h. The anhydrous sodium sulfate (700 g)was added, and the mixture was stirred for 1.5 h. The mixture wasfiltered, and the filter cake was washed with 1:1 heptane/ethyl acetate(2 L). The filtrate was concentrated under vacuum at <40° C. for 6 h.The resulting oil weighed 670 g (103% yield) and contains traces ofheptane, but is otherwise identical to previously prepared samples of 6,by NMR analysis.

Example 7 tert-butyl(R)-3-(2-(methylsulfonyloxy)ethyl)pyrrolidine-1-carboxylate (7)

Procedure A:

To a solution of tert-butyl(R)-3-(2-hydroxymethyl)pyrrolidine-1-carboxylate (41.0 g, 190 mmol)) wasadded triethylamine (40 mL) in toluene (380 mL) and cooled to −10° C.Methanesulfonyl chloride (20.0 mL, 256 mmol) was added slowly so as tomaintain the temperature around −5 to 0° C. The solution was warmed toambient temperature and stirred for 1 h. The solution was sampled hourlyand analyzed by HPLC to establish completion of the reaction. Uponcompletion of reaction, the solution was filtered, and the filtrate waswashed with a 5% sodium bicarbonate solution (250 mL). The organic layerwas collected and washed with a saturated aqueous sodium chloridesolution (250 mL). The organic layer was collected, dried over silicagel (10 g), and concentrated under vacuum to give 7 (53.0 g, 92.8%) as alight-yellow viscous oil. ¹H NMR (CDCl₃, 400 MHz) δ 4.26 (t, J=6.8 Hz,2H), 3.41-3.63 (m, 2H), 3.27 (m, 1H), 3.02 (s, 3H), 2.92 (m, 1H), 2.28(m, 1H), 2.05 (m, 1H), 1.83 (m, 2H), 1.50-1.63 (m, 1H), 1.46 (s, 9H).

Procedure B:

Under a nitrogen atmosphere, a solution of triethylamine (460 g, 4.55mol) and tert-butyl (R)-3-(2-hydroxymethyl)pyrrolidine-1-carboxylate(the entire sample from Example 7, Procedure B, 3.03 mol) in toluene(5.20 kg) was stirred and cooled to 5° C. Methanesulfonyl chloride (470g, 4.10 mol) was added slowly, over a 1.25 h, keeping the temperaturebelow 15° C. using ice bath cooling. The mixture was gradually warmed(over 1.5 h) to 35° C., and this temperature was maintained for 1.25 h,at which point GC analysis indicated that the reaction was complete. Themixture was cooled to 25° C., and solids were filtered off and thefilter cake washed with toluene (1.28 kg). The filtrate was stirred with10% aqueous sodium bicarbonate (4.0 kg) for 15 min, and the phases wereallowed to separate for 30 min. The organic phase was then stirred withsaturated aqueous sodium chloride (3.9 kg) for 30 min, and the phaseswere allowed to separate for 20 min. The organic phase was combined withsilica gel (160 g) and stirred for 1 h. Anhydrous sodium sulfate (540 g)was added, and the mixture was stirred an additional 40 min. The mixturewas then filtered, and the filter cake was washed with toluene (460 g).The filtrate was concentrated under vacuum at 50° C. for 5 h, and theresulting oil was kept under vacuum at 23° C. for an additional 8 h.This left 798 g of 7, 93% pure by GC analysis.

Example 8 Synthesis of tert-butyl (R)-3-vinylpyrrolidine-1-carboxylate(9)

Procedure A:

A solution of tert-butyl(R)-3-((methylsulfonyloxy)ethyl)pyrrolidine-1-carboxylate (49.0 g, 167mmol), sodium iodide (30.0 g, 200 mmol) and 1,2-dimethoxyethane (450 mL)was stirred at 50-60° C. for 4 h. The solution was sampled hourly andanalyzed by HPLC to establish completion of the reaction. Uponcompletion of reaction, the solution was cooled to −10° C., and solidpotassium tert-butoxide (32.0 g, 288 mmol) was added while maintainingtemperature below 0° C. The reaction mixture was warmed to ambienttemperature and stirred for 1 h. The mixture was sampled hourly andanalyzed by HPLC to establish completion of the reaction. Uponcompletion of reaction, the mixture was filtered through a pad ofdiatomaceous earth (25 g dry basis). The cake was washed with1,2-dimethoxyethane (100 mL). The combined filtrates were concentratedunder vacuum, to yield an orange oil with suspended solids. The oil wasdissolved in hexanes (400 mL), stirred for 30 min, and filtered toremove the solids. The organic layer was dried over silica gel (10 g),and concentrated under vacuum to give 9 (26.4 g, 82.9%) as a colorlessoil. ¹H NMR (CDCl₃, 400 MHz) δ 5.77 (m, 1H), 5.10 (dd, J=1.2 Hz, J=16Hz, 1H), 5.03 (dd, J=1.2 Hz, J=8.8 Hz, 1H), 3.41-3.59 (m, 2H), 3.29 (m,1H), 3.05 (m, 1H), 2.78 (m, 1H), 2.01 (m, 1H), 1.62-1.73 (m, 1H), 1.46(m, 9H).

Procedure B:

A solution of tert-butyl(R)-3-(2-(methylsulfonyloxy)ethyl)pyrrolidine-1-carboxylate (792 g ofthe product of Example 7, Procedure B, ˜2.5 mol), sodium iodide (484 g,3.27 mol) and 1,2-dimethoxyethane (7.2 L) was stirred at 55° C. for 4.5h under nitrogen, at which time GC analysis indicated that the reactionwas complete. The solution was cooled to <10° C., and solid potassiumtert-butoxide (484 g, 4.32 mol) was added in portions (1.25 h additiontime) while maintaining temperature below 15° C. The reaction mixturewas stirred 1 h at 5° C., warmed slowly (6 h) to 20° C., and stirred at20° C. for 1 h. The solution was filtered through a pad of diatomaceousearth (400 g dry basis). The filter cake was washed with1,2-dimethoxyethane (1.6 kg). The combined filtrates were concentratedunder vacuum, and the semisolid residue was stirred with heptane (6.0 L)for 2 h. The solids were removed by filtration (the filter cake waswashed with 440 mL of heptane), and the filtrate was concentrated undervacuum at 20° C. to give 455 g of 9 (90.7% pure). A sample of thismaterial (350 g) was fractionally distilled at 20-23 torr to give 296 gof purified 9 (bp 130-133° C.) (>99% pure by GC analysis).

Example 9 Synthesis of 3-bromo-5-(tetrahydro-2H-pyran-4-yloxy)pyridine(12)

A solution of 5-bromopyridin-3-ol (146 g, 834 mmol),tetrahydro-2H-pyran-4-ol (128 g, 1250 mmol), and triphenylphosphine (329g, 1250 mmol) in toluene (2.0 L) was heated to reflux, and 750 mL ofdistillate was removed via a Dean-Stark trap. The reaction mixture wascooled to 60° C., and 547 g (1.25 mol) of a 40% (w/w) solution of DEADin toluene was added drop-wise over a 1 hour period. The addition wasexothermic with the reactor temperature at the end of addition near 95°C. The reaction mixture was stirred at 115° C. for 18 h, and a portionof the reaction solution was sampled and analyzed by HPLC to establishthat the reaction was complete. Upon completion of reaction, 500 mL ofsolvent was removed by distillation, and the pot residue was cooled toambient temperature. This organic layer was washed with 10% aqueoussodium hydroxide (2×0.50 L) and concentrated under vacuum to produce aviscous oil, which was dissolved in 2N hydrochloric acid (1.0 L).Diatomaceous earth (100 g) was added with stirring and the resultingsuspension was filtered. The pad was rinsed with 2N hydrochloric acid(1.0 L), and the filtrates were combined and extracted with diisopropylether (500 mL). The diisopropyl ether layer was discarded, and theaqueous layer was treated with carbon black (10 g) and stirred at 45-50°C. for 1 h. The suspension was filtered through a pad of diatomaceousearth (25 g). The filtrate was collected, cooled to 5° C., and the pHadjusted with 50% aqueous sodium hydroxide (250 mL) to pH=13. Thesolution was extracted twice with chloroform (1.0 L, 600 mL), and thechloroform extracts were combined and concentrated under vacuum to give12 as a dark red viscous oil/low melting solid (187 g, 87%), which wasused without further purification. ¹H NMR (CDCl₃, 400 MHz) δ 8.29 (s,1H), 8.24 (s, 1H), 7.38 (s, 1H), 4.52 (m, 1H), 3.98 (m, 2H), 3.60 (m,2H), 2.05 (m, 2H), 1.81 (m, 2H).

Example 10 Synthesis of tert-butyl(R)-(E)-3-(2-(5-(tetrahydro-2H-pyran-4-yloxy)pyridin-3-yl)vinyl)pyrrolidine-1-carboxylate(13)

A mixture of tert-butyl (R)-3-vinylpyrrolidine-1-carboxylate 9 (7.00 g,35.5 mmol), 3-bromo-5-(tetrahydro-2H-pyran-4-yloxy)pyridine 12 (10.0 g,38.8 mmol), palladium acetate (0.40 g, 1.8 mmol), tricyclohexylphosphine(1.0 g, 3.57 mmol) and diisopropylethylamine (15 mL) in1-methyl-2-pyrrolidinone (130 mL) was stirred at 130° C. for 17 h. Thereaction was cooled to ambient temperature, diluted with water (800 mL)and extracted with ethyl acetate (2×200 mL). The combined organicextracts were dried over sodium sulfate, concentrated, and purified bysilica gel column chromatography using 60-100% ethyl acetate in hexanes.This product was further purified on reverse phase HPLC using 0.05%trifluoroacetic acid in acetonitrile and 0.05% trifluoroacetic acid inwater to obtain tert-butyl(R)-(E)-3-(2-(5-(tetrahydro-2H-pyran-4-yloxy)pyridin-3-yl)vinyl)pyrrolidine-1-carboxylate(11.0 g) as a gum.

¹H-NMR (CDCl₃, 400 MHz): δ 8.41 (s, 1H), 8.37 (d, J=2.3 Hz, 1H), 7.68(s, 1H), 6.48 (d, J=16.1 Hz, 1H), 6.43 (dd, J=16.0, 6.4 Hz, 1H),4.71-4.66 (m, 1H), 4.02-3.96 (m, 2H), 3.68-3.52 (m, 4H), 3.44-3.34 (m,1H), 3.28-3.15 (m, 1H), 3.09-2.98 (m, 1H), 2.18-2.04 (m, 3H), 1.90-1.78(m, 3H), 1.48 (s, 9H)

Example 11 Synthesis of(R)-3-(E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridine(14) hemigalactarate

A solution of tert-butyl(R)-(E)-3-(2-(5-(tetrahydro-2H-pyran-4-yloxy)pyridin-3-yl)vinyl)pyrrolidine-1-carboxylate(18 g, 48.13 mmol) in dichloromethane (40 mL) and trifluoroacetic acid(40 mL) was stirred at ambient temperature for 2 h. The reaction wasconcentrated on a rotary evaporator, and the residue was partitionedbetween saturated sodium chloride (50 mL) and chloroform (100 mL). Themixture was basified to pH 9 with 10% aqueous sodium hydroxide solution.The organic layer was separated and the aqueous layer extracted withchloroform (2×100 mL). The combined organic layers were dried oversodium sulfate and concentrated on a rotary evaporator to give(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydro-2H-pyran-4-yloxy)pyridine(8.0 g) as a gum. This was dissolved in methanol (100 mL) and galactaricacid (3.0 g, 14.6 mmol) was added and the mixture was heated to reflux.This hot solution was filtered, and filtrate was allowed to cool toambient temperature. The crystallized product was filtered and solid wassuspended in 10% water in ethanol (180 mL). The suspension was heated toreflux and hot solution was filtered. The filtrate was allowed to coolto ambient temperature. Crystallized product was filtered and dried onhigh vacuum pump to give(R)-3-((E)-2-pyrrolidin-3-ylvinyl)-5-(tetrahydro-2H-pyran-4-yloxy)pyridinehemigalactarate (4.5 g). MP: 179° C. ¹H-NMR (CD₃OD, 300 MHz): δ 8.04 (s,1H), 8.01 (d, J=2.2 Hz, 1H), 7.36 (s, 1H), 6.46 (d, J=16.0 Hz, 1H), 6.21(dd, J=16.0, 7.5 Hz, 1H), 4.65-4.4.54 (m, 1H), 4.12 (s, 1H), 3.89-3.83(m, 2H), 4.80 (s, 1H), 3.56-3.33 (m, 4H), 3.27-3.18 (m, 1H), 3.12-2.96(m, 2H), 2.23-2.14 (m, 1H), 1.98-1.91 (m, 2H), 1.88-1.78 (m, 1H),1.68-1.58 (m, 2H); MS (m/z): 275 (M+1).

Example 12 Large Scale Synthesis of(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridine(14) mono-L-malate

Under a nitrogen atmosphere, a mixture of3-bromo-5-(tetrahydro-2H-pyran-4-yloxy)pyridine (125 g of 85% purity,410 mmol), (R)-tert-butyl-3-vinylpyrrolidine-1-carboxylate (67.4 g, 340mmol), palladium(II) acetate (8.1 g, 36 mmol), tri-n-butylphosphine (15g, 74 mmol), potassium carbonate (74.0 g, 530 mmol), and DMAC (0.85 L)was stirred and heated at 130° C., monitoring for completion of reactionby LCMS. Upon completion of reaction, the reaction mixture was cooled toambient temperature and filtered through a pad of diatomaceous earth (50g dry basis), washing the pad with diisopropyl ether (0.60 L). Thefiltrate was combined with diisopropyl ether (0.60 L) and de-ionizedwater (0.50 L) and mixed for 15 min. The phases were allowed to separate(15 min), and the organic phase was collected. The aqueous phase wasextracted with a second portion of diisopropyl ether (0.60 L), using 15min of mixing and 15 min of settling time. The combined diisopropylether layers were washed with deionized water (2×0.50 L) andconcentrated under reduced pressure to produce a dark red viscous oil(136 g). This oil was dissolved in diisopropyl ether (1.40 L) and cooledto around 10° C. with an ice bath before charging 6N hydrochloric acid(0.40 L) via a dropping addition funnel over a 15 min period, keepingthe temperature below 20° C. The biphasic mixture was warmed to ambienttemperature (off-gassing occurred as it warmed) and stirred until LCMSindicated that the reaction was complete. Upon completion of reaction,the phases were allowed to separate, and the organic layer wasdiscarded. The pH of the aqueous layer was adjusted to pH 5-6 using 10%aqueous sodium hydroxide (0.485 L) and extracted with chloroform (0.25L). The chloroform layer was discarded. The aqueous layer was thenadjusted to pH>13 using 10% aqueous sodium hydroxide (0.075 L) and againextracted with chloroform (0.50 L). The chloroform extract wasconcentrated under reduced pressure to yield a red, viscous oil (55.0g). This material was a mixture of the desired(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-tetrahydro-2H-pyran-4-yloxy)pyridine(˜75%) and the corresponding Z (˜5%) and “exo” (˜20%) isomers by NMRanalysis. This result was reproducible over multiple runs.

The Z and “exo” impurities were removed from the desired(R)-3-((E)₂-(pyrrolidin-3-yl)vinyl)-5-tetrahydro-2H-pyran-4-yloxy)pyridineby conversion to the oxalate salt. A solution of oxalic acid (53.2 g.591 mmol) in a mixture of 2-propanol (0.20 L) and de-ionized water (0.09L) was prepared by stirring and heating at 50-55° C. (15 min). Thissolution was added, over a 5 min period, to a stirred solution of(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-tetrahydro-2H-pyran-4-yloxy)pyridine(82.0 g of 76% pure by HPLC, 299 mmol) in 2-propanol (1.0 L) held at70-75° C. The oxalic acid addition produced an exotherm (4-5° C.) thatwas controlled by adjusting the rate of addition. The heating source wasremoved and the solution was cooled slowly to 45-50° C. over 45 min. Aprecipitate formed quickly, beginning around 65-70° C. and becomingheavier as the resulting suspension cooled. The solids were collected byfiltration at 45-50° C. and washed successively with 2-propanol (2×0.25L) and hexanes (2×0.20 L). The tan solid was air dried for 2 h, afterwhich it weighed (95 g). NMR analysis indicated that the content of theZ and “exo” impurities had each been reduced to <1%. This result wasreproducible over multiple runs. Material of even greater purity wasobtained by recrystallization from ethanol/water. The stoichiometry ofthe salt was 2.3:1 acid/base (see Example 15).

A solution of(R)-3-((E)-2-(pyrrolidinium-3-yl)vinyl)-5-(tetrahydro-2H-pyran-4-yloxy)pyridiniumoxalate (380 g) in de-ionized water (2.6 L) was stirred and cooled toaround 10° C. with an ice bath. Aqueous sodium hydroxide (0.40 L of 25%)was added over a period of 15 min, keeping the temperature below 30° C.Chloroform (1.6 L) was then added, and the mixture was stirredvigorously for 20 min and filtered to remove insoluble sodium oxalate.The layers were allowed to separate, and the chloroform layer wascombined with Silicycle Si-Thiol® (21.6 g). The mixture was stirred andheated at 50-55° C. for 3-4 h, cooled to ambient temperature andfiltered. The filtrate was concentrated under reduced pressure toproduce a light red viscous oil (221 g). A portion of this free base(216 g) was dissolved in 2-propanol (1.2 L), heated to 70-75° C. andtreated with solid L-malic acid (106 g), using a 2-propanol rinse (100mL) to aid transfer. Dissolution of the solid produced an exotherm of5-7° C. over 3-5 min. The mixture was kept at 75-78° C. for 10 min, toensure complete dissolution of the solids, and then cooled slowly toambient temperature (90 min). As the temperature approached 65° C., thesolution was seeded with a few crystals of(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydro-2H-pyran-4-yloxy)pyridinemono-L-malate salt. After stirring at ambient temperature for 1 h, thesuspension was filtered. The collected solids were washed with2-propanol (2×0.80 L), air dried for 30 minutes, and vacuum dried at 78°C. for 8 h. The resulting off-white material weighed 297 g and was >99%pure by HPLC.

Example 13 Procedure for Screening for Salt Forms of(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridine

Test tubes (4 mL), provided with magnetic stir bars, were charged withequi-milimolar amounts of(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridinebase and the acid of interest (neat) and dissolved with 500 μL of either2-propanol or acetonitrile with the aid of heat. If precipitation didnot occur upon cooling, isopropyl acetate (100 μL) was added as ananti-solvent.

If no precipitation occurred, the solvent was evaporated under anitrogen stream with moderate heating and an alternative solvent wastried. Alternative solvents included acetone, ethyl acetate, isopropylacetate, absolute ethanol, acetonitrile, hexane, tert-butanol,tert-butyl acetate, and blends thereof.

The use of alcohols was avoided in the case of sulfonic acids. Isopropylacetate was used cautiously, while the use of acetone and ethyl acetatewas discontinued due to demonstrated reactivity of the secondary aminefunction of(R)-3-((E)-2-pyrrolidin-3-ylvinyl)-5-(tetrahydro-2H-pyran-4-yloxy)pyridinewith these solvents.

Results of these experiments are summarized in Table 1.

TABLE 1 Acids used in preliminary salt screen Acid Result4-Acetamidobenzoic oil Adipic oil (1 R,3S)-(+)-Camphoric oil (1 S)-(+)-1O-Camphorsulfonic oil Citric tacky gum Fumaric oil D-glucuronic browngum Hydrochloric red oil 4-Hydroxybenzoic tacky gum1-Hydroxy-2-naphthoic brown gum (Xinafoic) Maleic Oil L-Malic CrystalsMalonic Oil (R)-Mandelic Oil (S)-Mandelic Oil Methanesulfonic Oil4-Methoxybenzoic Oil Phosphoric Gum Succinic red oil L-Tartaric Oilp-Toluenesulfonic•H20 Oil

As demonstrated in Table 1, finding solid salt forms for(R)-3-((E)-2-pyrrolidin-3-ylvinyl)-5-(tetrahydro-2H-pyran-4-yloxy)pyridinewas challenging. Reported below are examples of solids salts and theirsyntheses.

Example 14 Preparation of(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridinemono-L-malate

To a stirred solution of(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridine(900 mg; 3.28 mmol) in 2-propanol (5 mL), heated to near boiling, wasadded L-malic acid (439.8 mg; 3.28 mmol, neat) in three portions. Thesolution was stirred near boiling for 10 min. Isopropyl acetate (1 mL)was added, heating was discontinued, and the solution was seeded whilestill hot. The solution was allowed to cool to ambient temperature (22°C.) with stirring whereupon the salt precipitated as a white granularsolid. The salt was re-dissolved by heating, re-seeded while hot, cooledand allowed to stand at ambient temperature without stirring for 24 h.The resulting plate-like crystals were collected by suction filtration,washed with isopropyl acetate (5 mL), and dried under nitrogen for 10min.

Further drying in vacuum oven at 70° C. for 1.5 h afforded 1.267 g(94.6%) of light-yellow crystals (m.p.=118-119° C.). ¹H-NMR (D₂O ord₆-DMSO) is consistent with a 1:1 acid:base stoichiometry. DSC exhibitsa single endotherm with maxima at 119.62° C. DVS shows minimum wateruptake up to 80% R.H. ¹H-NMR (D20, 400 MHz): δ 8.15 (s, 1H), 8.10 (s,1H), 7.58 (s, 1H), 6.52 (d, 1H), 6.28 (dd, 1H), 4.63 (m, 1H, partiallymasked by residual H2O resonance), 4.22 (dd, 1H), 3.88 (m, 2H), 3.55 (m,2H), 3.46 (dd, 1H), 3.38 (m, 1H), 3.25 (m, 1H), 3.11 (m, 1H), 3.02 (m,1H), 2.65 (dd, 1H), 2.42 (dd, 1H), 2.20 (m, 1H), 1.96 (m, 2H), 1.85 (m,1H), 1.68 (m, 2H). ¹H-NMR (d₆-DMSO, 400 MHz): δ 8.20 (s, 1H), 8.18 (s,1H), 7.52 (s, 1H), 6.55 (d, 1H), 6.46 (dd, 1H), 4.68 (m, 1H), 3.87 (m,3H), 3.49 (m, 2H), 3.40 (dd, 1H), 3.32 (m, 1H), 3.18 (m, 1H), 3.05 (m,1H), 2.93 (m, 1H), 2.51 (dd, 1H, partially masked by residual DMSO),2.31 (dd, 1H), 2.14 (m, 1H), 1.98 (m, 2H), 1.80 (m, 1H), 1.58 (m, 2H).

Example 15 Preparation of(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineoxalate

A warm solution of (1.137 g of 85%, 3.52 mmol corrected for purity) in2-propanol (8.5 mL) and water (0.4 mL) was treated with oxalic acid(0.373 g, 4.14 mmol), as a solid, in one portion. The resulting mixturewas stirred and heated to near reflux. A few solids began to precipitatefrom the hot solution. The mixture was allowed to cool to ambienttemperature. The off-white solids were filtered (Büchner), washed with2-propanol (10 mL, 8 mL) and dried under vacuum (with an air bleed) at50° C. for 3 h to yield 0.861 g (50.8% yield based upon a 2.3 oxalatestoichiometry, corrected for purity of the starting material) of anoff-white powder. A 0.765 g sample of this material was recrystallizedfrom a mixture of 2-propanol (8 mL) and water (1.3 mL), heated atreflux. Upon cooling to ambient temperature, the resulting solids werefiltered (Büchner), washed with 2-propanol (10 mL), dried under vacuum(with an air bleed) at 50° C. for 4 h and then further vacuum dried withan air bleed) at 70° C. for 24 h to afford 0.441 g (57.6% recovery) ofan off-white to white solid, mp 180-181° C.

Cacl'd for C₁₆H₂₂N₂O₂. 2.3 C₂H₂O₄: C, 51.39; H, 5.57; N, 5.82. Found: C,51.09, 51.24; H, 5.67, 5.66; N, 5.84, 5.92.

¹H-NMR (D₂O, 400 MHz) δ 8.23 (s, 1H), 8.20 (s, 1H), 7.96 (s, 1H), 6.54(d, 1H), 6.40 (dd, 1H), 4.73 (m, 1H), 3.84 (m, 2H), 3.54 (m, 2H), 3.45(dd, 1H), 3.35 (m, 1H), 3.23 (m, 1H), 3.12 (m, 1H), 3.02 (m, 1H), 2.16(m, 1H), 1.96 (m, 2H), 1.83 (m, 1H), 1.68 (m, 2H).

Example 16 Preparation of(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridinedi-p-toluoyl-D-tartrate

To a stirred solution of(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridine(4.0 g, 15 mmol) in ethanol (12.5 mL) solution, heated to 60° C., wasadded solid di-p-toluoyl-D-tartaric acid (5.3 g, 14 mmol). The solutionwas held at 60° C. for 2-3 min to ensure complete dissolution of thesolids, then the heat source was removed and the solution was cooled to25-30° C. over 60 min. The resulting suspension was held at 25-30° C.for 30 min, and then filtered to collect the solids. The solids werewashed with ethanol (2×20 mL), air dried for 30 min, then dried in avacuum oven under reduced pressure at 50° C., until a constant weightwas established, to give(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridinedi-p-toluoyl-D-tartrate as an off-white solid (6.7 g, 72%). NNNMRanalysis indicated a 1:1 salt stoichiometry. ¹H-NMR (DMSO-d₆, 400 MHz) δ8.18 (s, 1H), 8.15 (s, 1H), 7.86 (d, 4H), 7.47 (s, 1H), 7.32 (d, 4H),6.43 (d, 1H), 6.36 (m, 1H), 5.67 (s, 2H), 4.69 (m, 1H), 3.85 (m, 2H),3.49 (m, 2H), 3.25 (m, 2H), 3.10 (m, 1H), 2.88 (m, 2H), 2.39 (s, 6H),1.98 (m, 3H), 1.60 (m, 3H).

Biological Assays Example 17 Radioligand Binding at CNS nAChRs: α4β2 NNRSubtype

Preparation of Membranes from Rat Cortex:

Rats (female, Sprague-Dawley), weighing 150-250 g, were maintained on a12 h light/dark cycle and were allowed free access to water and foodsupplied by PMI Nutrition International, Inc. Animals were anesthetizedwith 70% CO₂, and then decapitated. Brains were removed and placed on anice-cold platform. The cerebral cortex was removed and placed in 20volumes (weight:volume) of ice-cold preparative buffer (137 mM NaCl,10.7 mM KCl, 5.8 mM KH2PO₄, 8 mM Na₂HPO₄, 20 mM HEPES (free acid), 5 mMiodoacetamide, 1.6 mM EDTA, pH 7.4); PMSF, dissolved in methanol to afinal concentration of 100 μM, was added and the suspension washomogenized by Polytron. The homogenate was centrifuged at 18,000×g for20 min at 4° C. and the resulting pellet was re-suspended in 20 volumesof ice-cold water. After 60 min incubation on ice, a new pellet wascollected by centrifugation at 18,000×g for 20 min at 4° C. The finalpellet was re-suspended in 10 volumes of buffer and stored at −20° C.

Preparation of Membranes from SH-EP1/human α4β2 Clonal Cells:

Cell pellets from 40 150 mm culture dishes were pooled, and homogenizedby Polytron (Kinematica GmbH, Switzerland) in 20 milliliters of ice-coldpreparative buffer. The homogenate was centrifuged at 48,000 g for 20min at 4° C. The resulting pellet was re-suspended in 20 mL of ice-coldpreparative buffer and stored at −20° C.

Assay.

On the day of the assay, the frozen membranes were thawed and spun at48,000×g for 20 min. The supernatant was decanted and discarded. Thepellet was resuspended in Dulbecco's phosphate buffered saline (PBS,Life Technologies) pH 7.4 and homogenized with the Polytron for 6seconds. Protein concentrations were determined using a Pierce BCAProtein Assay Kit, with bovine serum albumin as the standard (PierceChemical Company, Rockford, Ill.).

Membrane preparations (approximately 50 μg for human and 200-300 μgprotein for rat αβ2) were incubated in PBS (50 μL and 100 μLrespectively) in the presence of competitor compound (0.01 nM to 100 μM)and 5 nM [³H]nicotine for 2-3 h on ice. Incubation was terminated byrapid filtration on a multi-manifold tissue harvester (Brandel,Gaithersburg, Md.) using GF/B filters presoaked in 0.33%polyethyleneimine (w/v) to reduce non-specific binding. Tissue wasrinsed 3 times in PBS, pH 7.4. Scintillation fluid was added to filterscontaining the washed tissue and allowed to equilibrate. Filters werethen counted to determine radioactivity bound to the membranes by liquidscintillation counting (2200CA Tri-Carb LSC, Packard Instruments, 50%efficiency or Wallac Trilux 1450 MicroBeta, 40% efficiency, PerkinElmer).

Data were expressed as disintegrations per minute (DPMs). Within eachassay, each point had 2-3 replicates. The replicates for each point wereaveraged and plotted against the log of the drug concentration. IC₅₀,which is the concentration of the compound that produces 50% inhibitionof binding, was determined by least squares non-linear regression. Kivalues were calculated using the Cheng-Prussof equation (1973):

Ki=IC ₅₀/(1+N/Kd)

where N is the concentration of [³H]nicotine and Kd is the affinity ofnicotine (3 nM, determined in a separate experiment).

Example 18 Radioligand Binding at CNS nAChRs: α7 NNR Subtype

Rats (female, Sprague-Dawley), weighing 150-250 g, were maintained on a12 h light/dark cycle and were allowed free access to water and foodsupplied by PMI Nutrition International, Inc. Animals were anesthetizedwith 70% CO₂, and then decapitated. Brains were removed and placed on anice-cold platform. The hippocampus was removed and placed in 10 volumes(weight:volume) of ice-cold preparative buffer (137 mM NaCl, 10.7 mMKCl, 5.8 mM KH2PO₄, 8 mM Na₂HPO₄, 20 mM HEPES (free acid), 5 mMiodoacetamide, 1.6 mM EDTA, pH 7.4); PMSF, dissolved in methanol to afinal concentration of 100 μM, was added and the tissue suspension washomogenized by Polytron. The homogenate was centrifuged at 18,000×g for20 min at 4° C. and the resulting pellet was re-suspended in 10 volumesof ice-cold water. After 60 min incubation on ice, a new pellet wascollected by centrifugation at 18,000×g for 20 min at 4° C. The finalpellet was re-suspended in 10 volumes of buffer and stored at −20° C. Onthe day of the assay, tissue was thawed, centrifuged at 18,000×g for 20min, and then re-suspended in ice-cold PBS (Dulbecco's PhosphateBuffered Saline, 138 mM NaCl, 2.67 mM KCl, 1.47 mM KH2PO₄, 8.1 mMNa₂HPO₄, 0.9 mM CaCl₂, 0.5 mM MgCl₂, Invitrogen/Gibco, pH 7.4) to afinal concentration of approximately 2 mg protein/mL. Protein wasdetermined by the method of Lowry et al., J. Biol. Chem. 193: 265(1951), using bovine serum albumin as the standard.

The binding of [³H]MLA was measured using a modification of the methodsof Davies et al., Neuropharmacol. 38: 679 (1999), herein incorporated byreference with regard to such method. [³H]MLA (Specific Activity=25-35Ci/mmol) was obtained from Tocris. The binding of [³H]MLA was determinedusing a 2 h incubation at 21° C. Incubations were conducted in 48-wellmicro-titre plates and contained about 200 μg of protein per well in afinal incubation volume of 300 μL. The incubation buffer was PBS and thefinal concentration of [³H]MLA was 5 nM. The binding reaction wasterminated by filtration of the protein containing bound ligand ontoglass fiber filters (GF/B, Brandel) using a Brandel Tissue Harvester atambient temperature. Filters were soaked in de-ionized water containing0.33% polyethyleneimine to reduce non-specific binding. Each filter waswashed with PBS (3×1 mL) at ambient temperature. Non-specific bindingwas determined by inclusion of 50 μM non-radioactive MLA in selectedwells.

The inhibition of [³H]MLA binding by test compounds was determined byincluding seven different concentrations of the test compound inselected wells. Each concentration was replicated in triplicate. IC₅₀values were estimated as the concentration of compound that inhibited 50percent of specific [³H]MLA binding. Inhibition constants (Ki values),reported in nM, were calculated from the IC₅₀ values using the method ofCheng et al., Biochem. Pharmacol. 22: 3099-3108 (1973).

Selectivity vs. Peripheral nAChR5 Example 19 Interaction at the HumanMuscle nAChR Subtype

Activation of muscle-type nAChRs was established on the human clonalline TE671/RD, which is derived from an embryonal rhabdomyosarcoma(Stratton et al., Carcinogen 10: 899 (1989)). These cells expressreceptors that have pharmacological (Lukas, J. Pharmacol. Exp. Ther.251: 175 (1989)), electrophysiological (Oswald et al., Neurosci. Lett.96: 207 (1989)), and molecular biological profiles (Luther et al., J.Neurosci. 9: 1082 (1989)) similar to the muscle-type nAChR.

TE671/RD cells were maintained in proliferative growth phase accordingto routine protocols (Bencherif et al., Mol. Cell. Neurosci. 2: 52(1991) and Bencherif et al., J. Pharmacol. Exp. Ther. 257: 946 (1991)).Cells were cultured in Dulbecco's modified Eagle's medium (Gibco/BRL)with 10% horse serum (Gibco/BRL), 5% fetal bovine serum (HyClone, LoganUtah), 1 mM sodium pyruvate, 4 mM L-Glutamine, and 50,000 unitspenicillin-streptomycin (Irvine Scientific). When cells were 80%confluent, they were plated to 12 well polystyrene plates (Costar).Experiments were conducted when the cells reached 100% confluency.

Nicotinic acetylcholine receptor (nAChR) function was assayed using⁸⁶Rb⁺ efflux according to the method described by Lukas et al., Anal.Biochem. 175: 212 (1988). On the day of the experiment, growth media wasgently removed from the well and growth media containing ⁸⁶Rubidiumchloride (10⁶ μCi/mL) was added to each well. Cells were incubated at37° C. for a minimum of 3 h. After the loading period, excess ⁸⁶Rb⁺ wasremoved and the cells were washed twice with label-free Dulbecco'sphosphate buffered saline (138 mM NaCl, 2.67 mM KCl, 1.47 mM KH2PO₄, 8.1mM Na₂HPO₄, 0.9 mM CaCl₂, 0.5 mM MgCl₂, Invitrogen/Gibco, pH. 7.4),taking care not to disturb the cells. Next, cells were exposed to either100 μM of test compound, 100 μM of L-nicotine (Acros Organics) or bufferalone for 4 min. Following the exposure period, the supernatantcontaining the released ⁸⁶Rb⁺ was removed and transferred toscintillation vials. Scintillation fluid was added and releasedradioactivity was measured by liquid scintillation counting.

Within each assay, each point had 2 replicates, which were averaged. Theamount of ⁸⁶Rb⁺ release was compared to both a positive control (100 μML-nicotine) and a negative control (buffer alone) to determine thepercent release relative to that of L-nicotine.

When appropriate, dose-response curves of test compound were determined.The maximal activation for individual compounds (Emax) was determined asa percentage of the maximal activation induced by L-nicotine. Thecompound concentration resulting in half maximal activation (EC50) ofspecific ion flux was also determined.

Example 20 Interaction at the Human Ganglionic nAChR Subtype

The cell line SH-SY5Y is a continuous line derived by sequentialsubcloning of the parental cell line, SK-N-SH, which was originallyobtained from a human peripheral neuroblastoma. SH-SY5Y cells express aganglion-like nAChR (Lukas et al., Mol. Cell. Neurosci. 4: 1 (1993)).

Human SH-SY5Y cells were maintained in proliferative growth phaseaccording to routine protocols (Bencherif et al., Mol. Cell. Neurosci.2: 52 (1991) and Bencherif et al., J. Pharmacol. Exp. Ther. 257: 946(1991)). Cells were cultured in Dulbecco's modified Eagle's medium(Gibco/BRL) with 10% horse serum (Gibco/BRL), 5% fetal bovine serum(HyClone, Logan Utah), 1 mM sodium pyruvate, 4 mM L-Glutamine, and50,000 units penicillin-streptomycin (Irvine Scientific). When cellswere 80% confluent, they were plated to 12 well polystyrene plates(Costar). Experiments were conducted when the cells reached 100%confluency.

Nicotinic acetylcholine receptor (nAChR) function was assayed using⁸⁶Rb⁺ efflux according to a method described by Lukas et al., Anal.Biochem. 175: 212 (1988). On the day of the experiment, growth media wasgently removed from the well and growth media containing ⁸⁶Rubidiumchloride (10⁶ μCi/mL) was added to each well. Cells were incubated at37° C. for a minimum of 3 h. After the loading period, excess ⁸⁶Rb+ wasremoved and the cells were washed twice with label-free Dulbecco'sphosphate buffered saline (138 mM NaCl, 2.67 mM KCl, 1.47 mM KH2PO₄, 8.1mM Na₂HPO₄, 0.9 mM CaCl₂, 0.5 mM MgCl₂, Invitrogen/Gibco, pH 7.4),taking care not to disturb the cells. Next, cells were exposed to either100 μM of test compound, 100 μM of nicotine, or buffer alone for 4 min.Following the exposure period, the supernatant containing the released⁸⁶Rb⁺ was removed and transferred to scintillation vials. Scintillationfluid was added and released radioactivity was measured by liquidscintillation counting

Within each assay, each point had 2 replicates, which were averaged. Theamount of ⁸⁶Rb⁺ release was compared to both a positive control (100 μMnicotine) and a negative control (buffer alone) to determine the percentrelease relative to that of L-nicotine.

When appropriate, dose-response curves of test compound were determined.The maximal activation for individual compounds (Emax) was determined asa percentage of the maximal activation induced by L-nicotine. Thecompound concentration resulting in half maximal activation (EC₅₀) ofspecific ion flux was also defined.

Example 21 Novel Object Recognition

Memory was assessed by using a three-trial novel object recognition test(Luine et al., Pharm. Biochem. Behay. 74, 213-220 (2002)). On the firstday (exploratory trial), rats were allowed to explore an open arena(44.5×44.5×30.5 cm) for 6 min. On the second day (acquisition trial),rats were allowed to explore the same arena in the presence of twoidentical objects (both object A) for 3 min. On the third day (retentionor recall trial), performance was evaluated by allowing the same animalto re-explore the arena for 3 min in the presence of two differentobjects: the familiar object A and a novel object B. An inter-trialinterval of 24 h was imposed between the three NOR trials. Recognitionmemory was assessed by comparing the time spent exploring a novel(object B) versus a familiar (object A) object during the recall trial.Recognition index was assessed for each animal and expressed as a ratio((time B/time A+time B)×100).

Example 22 Radial Arm Maze

Working memory was assessed in a radial arm maze (RAM) task. The RAMtask was conducted using an automated eight-arm maze (Med Associates,Inc.) The maze was located on a circular table approximately 88 cm abovethe floor with overhead lighting in a dedicated testing ambient andlarge, high contrast geometric shapes on the wall. Furthermore,additional visual cues were located at the hub entry into each arm,above each the food hopper and on the ceiling. The central platformmeasured 30.5 cm in diameter with eight arms (9 cm W×45.7 cm L×16.8 cmH) radiating from it. Automatic guillotine doors were located at theentrance to each runway with a pellet receptacle at the distal end ofeach arm. White noise will be audible during all training and testingprocedures. Activity on the maze was monitored by tracking quantitativeactivity (generated by infra-red beam breaks) on the computer interfaceand monitor screen.

Following the baseline assessment and after re-attainment test sessioncriterion, animals were assessed for their sensitivity tochemically-induced cognitive impairment using the muscarinic antagonistscopolamine (0.2-0.4 mg/kg; s.c.). A dose of scopolamine was determinedfor each animal based on the minimum dose that produced significant andreliable cognitive impairment. Scopolamine was administered 0.5 h priorto the acquisition phase trial whereas,(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridinehemi-galactarate (0.03 mg/kg; p.o.) was administered 0.5 h prior to thestart of the recall (or test) phase trial. In the acquisition trial, onerandomly selected arm was blocked with a Plexiglas barrier situated justinside the arm, behind the hub door. The animal was placed in thecentral hub of the maze with doors down. After approximately 10 sec,doors to the 7 available arms were raised. The first entry to each openarm was reinforced with a sucrose food pellet. The session ended afterall 7 available arms were visited or 5 min elapsed. The order of armsvisited, reinforcers received, errors (re-entries), time to complete thetask, the number of entries and time required to enter 7 available armsand consume food reinforcer were recorded. In the recall trial, all 8arms were available, however, only the first visit to the previouslyblocked arm (i.e., the arm that was blocked during the acquisitiontrial) was reinforced. The session ended once the previously blocked armwas visited and the reinforcer was consumed or 5 min elapsed. For therecall trial, re-entry errors, the number of (incorrect) arms enteredprior to choosing the arm that was blocked during the acquisition trialand the time taken to complete the trial was recorded. The delay betweenthe acquisition and test phase trials was 24 h.

Example 23 CYP Inhibition Studies

Inhibition of CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 catalyticactivity by(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineor a slat thereof was assessed using a fluorogenic CYP assay. Probesubstrates which fluoresce upon CYP catalyzed oxidation were used toevaluate the degree of inhibition of the test substrate. A singleconcentration of each probe substrate (at approximately the apparentK_(m) value) and two different concentrations of(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridinehemigalactarate (2 and 20 μM) were tested in duplicate. Fluorescenceintensity at selected wavelengths was used as a measure of enzymeactivity. Decreased fluorescence in the presence of(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineor a sat thereof was an indication of inhibition. Positive controls(known inhibitors) wlere run concurrently to demonstrate method controland CYP activity. Duplicate samples were run alongside the positive andnegative controls. Incubation of test samples was performed at 37° C.Experimental parameters are outlined in Table 2.

TABLE 2 Experiment conditions for fluorescence CYP inhibition assaysCYP1A2 CYP2C9 CYP2C19 CYP2D6 CYP3A4 Enzyme 1 2 2 3 1 amount (pmol/well)Probe 3 μM CEC 75 μM MFC 100 μM MFC 20 μM MAMC 15 μM BFC Substrate KPO4pH 7.4 0.1M 0.05M 0.05M 0.1M 0.1M NADPH conc. 1 mM 1 mM 1 mM 0.06 mM 1mM Incubation 20 min 50 min 40 min 35 min 30 min time Ex/Em λ (nm)405/460 405/530 405/530 390/460 405/530 Gain 20 40 30 10 50 ReferenceFurafylline Sulfaphenazole Tranylcypromine Quinidine Ketoconazoleinhibitor Expected 15~25 3~5 3~5 3~6 4~15 signal/noise IC50 (μM) for ~1~1 ~6 ~0.01 ~0.06 reference inhibitor CEC = 3-Cyano-7-ethoxy-coumarinMFC = 7-methoxy-4-trifluoromethyl-coumarin MAMC =7-methoxy-4-(aminomethyl)-coumarin BFC =7-bensyloxy-4-trifluoromethyl-coumarin

Summary of Biological Data

In vitro pharmacology

A summary of the in vitro primary pharmacology data for(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineor a salt thereof is presented in Table 3 and discussed in detail below.

Primary Pharmacology and Selectivity:

The capacity of(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineor a salt thereof to bind to α4β2 receptors was determined with receptorbinding inhibition assays using human recombinant α4β2 receptorsexpressed in SH-EP1 cellular membranes and rat native α4β2 receptors inrat cortical membranes.

(R)-3-((E)-2-(Pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineor a salt thereof inhibited the binding of [³H]-nicotine to humanrecombinant α4β2 nicotinic receptors with a K_(i) of 2 nM and[³H]epibatidine to rat native α4β2 receptors with a K_(i) of 4 nM.

(R)-3-((E)-2-(Pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineor a salt thereof inhibited the binding of [³H]methyllycaconitine (MLA)to rat native a7 receptors in rat hippocampal membranes with a K_(i)of >10000 nM.(R)-3-((E)-2-(Pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineor a salt thereof also displayed reduced affinity for human nativeganglion-type nicotinic receptors (likely α3β4), inhibiting the bindingof [³H]epibatidine to receptors in SH-SY5Y membranes with a K_(i) of3400 nM, and reduced affinity for human native muscle-type nicotinicreceptors (likely α1β1γδ), inhibiting the binding of [³H]epibatidine toreceptors in TE-671 membranes with a K_(i) of 25000 nM.

TABLE 3 Summary of(R)-3-(2-(pyrrolidin-3-yl)vinyl)-5-((tetrahydro-2H-pyran- 4yl)oxy)pyridine, or a salt thereof, in vitro pharmacology Targetaffinity and activation Rat cortex binding K_(i) 4 nM Human recombinant(SH-EP 1) α4β2 binding K_(i) 2 nM Rat hippocampus (α7_(.), K_(i) >10000nM Human ganglionic (SH-SY5Y), K_(i) 3400 nM Human (TE671/RD) muscle,K_(i) 25 μM Human recombinant (SH-EP 1) α4β2 EC₅₀, 0.1 μM, 76% Emax (Caflux) Human ganglionic (SH-SY5Y), EC₅₀, Emax (Ca 11 μM, 13% flux) Human(TE671/RD) muscle, EC₅₀, Emax (Ca 13 μM, 37% flux) Multiple receptorscreening assay Only nicotinic

Cellular Efficacy:

The aim of these studies was to determine functional activity of(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineor a salt thereof at human recombinant α4β2 receptors.(R)-3-((E)-2-(Pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineor a salt thereof is an α4β2 nicotinic agonist activating the receptorwith an EC₅₀ of 0.1 μM and an E_(max) of 76% in relation to 10 μMnicotine in a calcium flux assay with SH-EP1/human α4β2 cells following24-h incubation at 29° C.

(R)-3-((E)-2-(Pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineor a salt thereof was tested in ganglion and muscle-type nicotinicreceptor ion flux assays to examine functional selectivity. In Ca⁺⁺efflux assays,(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineor a salt thereof has an EC₅₀ of 11 μM and an E_(max) of 13% at humannative ganglion receptors in SH-SY5Y cells, an EC₅₀ of 13 μM and anE_(max) of 37% at human native muscle receptors in TE-671 cells.

In Vitro Secondary Pharmacology: Multiple Receptor Screening Assay

(R)-3-((E)-2-(Pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineor a salt thereof was tested for selectivity against a panel of 65receptors. At a single concentration of 10 μM,(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineor a salt thereof inhibited the binding of labelled ligand only toneuronal nicotinic receptors (α-BnTx insensitive) with 99% inhibition.

Inhibition of hERG

The IC₅₀ for the inhibition of hERG (human HEK-239 cells) by(R)-3-((E)-2-pyrrolidin-3-ylvinyl)-5-(tetrahydro-2H-pyran-4-yloxy)pyridineor a salt thereof was determined to be 84 μM.

In Vivo Pharmacology

(R)-3-((E)-2-(Pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineor a salt thereof improved long-term visual episodic/declarative memoryas assessed by novel object recognition (NOR) task following oral dosingin normal rats. The results of these studies are presented in FIG. 1.The recognition index of the vehicle-treated group 24 h after theacquisition trial was 50±0.5% demonstrating the inability of this groupto recognize the familiar object after this delay (left panel). Bycontrast, animals treated with(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineor a salt thereof exhibited recognition indexes of 71±2% at the 0.04μmol/kg dose level and 61±3% and the 1.1 μmol/kg dose level (leftpanel). In a follow-up NOR study (experimental procedures were identicalas used in the first NOR study), the minimum effect dose (MED) level for(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineor a salt thereof was determined to be 0.004 μmol/kg (right panel)suggesting that the rats are able to recognize the familiar object atall doses levels tested. In the two “recall only” sessions; subset ofanimals were orally dosed with water on day 1 (i.e., exploratorysession) and day 2 (i.e., acquisition session) and then orally dosedeither with 1.1 μmol/kg(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineor a salt thereof (left panel) or 0.04 μmol/kg (right panel) on day 3(i.e., recall session). Even following a single oral administration,(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineor a salt thereof demonstrated pro-cognitive effects at these two doselevels. At both dose levels,(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineor a salt thereof exhibited recognition indexes significantly abovecontrols, indicating recognition of the familiar object following acutedosing. In the Figure, the dashed line at 65% denotes subjectivecriteria for biological cognitive enhancing activity. *P<0.05.

(R)-3-((E)-2-(Pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineor a salt thereof was evaluated for its duration of effect in the NORtask in normal rats. The results of these studies are presented in FIG.2. The recognition index of the vehicle-treated group at 0.5 h followingdosing on the recall trial was 52±0.8% demonstrating the inability ofthis group to recognize the familiar object after this delay. Bycontrast, animals treated with(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineor a salt thereof (0.004 μmol/kg: oral) exhibited recognition indexes of72±2% at 0.5 h, 70±3% at 6 h and 70±4% at 8 h suggesting that rats areable to recognize the familiar object for up to 8 h after dosing. In theFigure, the dashed line at 65% denotes subjective criteria forbiological cognitive enhancing activity (*P<0.05).

On the basis of these studies, a likely pharmacological effect ispossible when dosing(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineor a salt thereof over a wide range, including relatively low doselevels. One embodiment of the present invention relates to dosing(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineor a pharmaceutically acceptable salt thereof in oral doses as low asabout 0.004 μmol/kg. One embodiment of the present invention relates toan oral dose of less than 100 mg, preferably less than 50 mg, morepreferably less than 10 mg, and most preferably less than 1 mg. Theseeffective doses typically represent the amount administered as a singledose, or as one or more doses administered over a 24 h period.

Radial Arm Maze (RAM) Studies

In a second cognitive assay,(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineor a salt thereof attenuated cognitive deficits, induced by scopolamine,in an animal model of working memory. Results of these experiments areillustrated in FIG. 3. During the acquisition trial, rats were allowedaccess to 7 of the eight arms whereas, in the test trial, all 8 armswere available, however, only the first visit to the previously blockedarm (i.e., the arm that was blocked during the acquisition trial) wasreinforced. Scopolamine (0.3±0.1 mg/kg; s.c.) was administered 0.5 hprior to the acquisition trial, and(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineor a salt thereof (0.03 mg/kg or 0.1 μmol/kg; p.o.) was administered 0.5h prior to the test trial.(R)-3-((E)-2-(Pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineor a salt thereof was able to reverse scopolamine-induced cognitivedeficits (*P<0.05).

Human Cytochrome P450 (CYP) Inhibition, Induction, Transport, andDrug-Drug Interaction Potential

A CYP450 inhibition assay using fluorescent substrates and recombinantenzymes showed no evidence of inhibition by(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineor a salt thereof of the 5 major CYPs (IC₅₀>20 μM, Table 4). Inaddition, no evidence of time-dependent inhibition of CYP3A4, CYP2D6,CYP2B6, CYP2C9, or CYP1A2 was observed. No PXR (pregnane X receptor)activation by(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridinewas observed up to 10 μM, thus the risk related to induction of P450s isbelieved to be negligible.

TABLE 4 CYP inhibition IC₅₀ (μM) CYP mediated metabolism 1A2 >20 3A4 >202C9 >20 2C19 >20 2D6 >20

(R)-3-((E)-2-(Pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineor a salt thereof exhibits low hepatic turnover rate in human livermicrosomes or hepatocytes. Preliminary phenotyping data suggested thatboth CYP2D6 and FMO3 contributed to the metabolism of(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineor a salt thereof. In addition, renal clearance was expected to be themajor elimination route, contributing more than 50% of the totalclearance in human. Therefore any variation of(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridineor a salt thereof metabolism in human due to CYP polymorphism isexpected to be less than 2 fold due to the significant renal clearanceand low hepatic clearance.

Test compounds for the experiments described herein were employed infree or salt form, and, if not otherwise stated, the test compound is(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridinehemigalactarate.

The specific pharmacological responses observed may vary according toand depending on the particular active compound selected or whetherthere are present pharmaceutical carriers, as well as the type ofpharmaceutical composition and mode of administration employed, and suchexpected variations or differences in the results are contemplated inaccordance with practice of the present invention.

Although specific embodiments of the present invention are hereinillustrated and described in detail, the invention is not limitedthereto. The above detailed descriptions are provided as exemplary ofthe present invention and should not be construed as constituting anylimitation. Modifications will be obvious to those skilled in the art,and all modifications that do not depart from the spirit of theinvention are intended to be included with the scope of the appendedclaims.

What is claimed is:
 1. An administration regimen of a pharmaceuticalcomposition comprising administering(R)-3-((E)-2-(pyrrolidin-3-yl)vinyl)-5-(tetrahydropyran-4-yloxy)pyridine,or a pharmaceutically acceptable salt thereof in amounts of between 7 to2200 μg/kg.